Immunohistochemistry Seminar 5

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IHC

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GOOD MORNING:

GOOD MORNING

Immuno histo chemistry:

Immuno histo chemistry Presented by: Dr.Sourab Kumar Guided By: Dr.Avinash Tamgadge (HOD) Dr. BhaleRao (Professor) Dr. Sandhya Tamgadge (Professor) Dr.D.Y.Patil Dental College Oral Pathology & Microbiology

Index:

Index Basic immunohistochemistry steps Definition Antigens IgG antibodies Monoclonal antibodies formation Polyclonal antibodies formation Handling of antibodies

Index(Continued):

Index(Continued) Basic Enzymology Labels Antigen-antibody binding reaction Direct Method Indirect Method Antigen Retrieval Techniques Blocking Endogenous Enzymes Blocking Background Staining

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IMMUNOHISTOCHEMISTRY STEPS Slide 3 of 23 Tissue sections Antigen retrieval Blocking endogenous enzymes Secondary antibody Primary antibody Microscopy Observation Blocking background staining Chromogen Substrate Counterstain Mounting

Antigen retrieval:

Antigen retrieval Routine histology procedures masks the antigens, usually by formalin cross-linking, or even destroys some antigen epitopes.So to retrieve this antigens we use retrieval procedures.

Blocking endogenous enzymes:

Blocking endogenous enzymes Most popular enzymes used today are trypsin and protease. Because digestion breaks down formalin cross-linking and hence the antigenic sites for a number of antibodies are uncovered. Impurities in crude trypsin such as chymotrypsin are the active ingredients.Essential that commercial companies constantly monitor & standardise chymotrypsin levels in the batches of trypsin they supply in order to assist users with the reproducility of their technique. Enzymes similar to those used as tracer are present in tissue, they may react with the substrate used to localize the tracer and give rise to problems in interpretation.

Contd.:

Contd. Peroxidase and substances giving a pseudo-peroxidase reaction are present in some normal and neoplastic tissues i.e. leucocytes and erythocytes. Endogenous peroxidases are first localised by using one chromogen and then following immunocytochemical staining, the enzyme tracer is localized by an alternative chromogen which yields a reaction end-product in contrasting color.

Blocking background staining:

Blocking background staining Non-specific uptake of antigen, particularly high affinity of collagen and reticulin for immunoglobulins can cause high levels of background staining. Specific background staining also occurs when primary antibody is contaminated with other antibodies. Non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections

A.H.Coons:

A.H.Coons In 1940, (Microbiologist) described identification of tissue antigens in pneumococci by using direct fluorescence

Definitions:

Definitions Immunohistochemistry is a technique for identifying tissue constituents (antigens) by means of antigen-antibody interactions. The site of antibody binding being identified either by direct labeling of the antibody, or by use of secondary antibody labeling method .

Is Immunohistochemistry(IHC) and Immunocytochemistry(ICC) one and the same?:

Is Immunohistochemistry(IHC) and Immunocytochemistry(ICC) one and the same?

Mobility of Antigens:

Mobility of Antigens

IgG Antibodies:

IgG Antibodies VL and VH together form the antigen-combining site. The H chain also has three constant domains (CH1, CH2,CH3) and carries the carboxyl terminal portion of the immunoglobulin.

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Digestion by papain results in the cleavage of a susceptible bond on the N-terminal side of the inter-heavy chain disulfide bridges .

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Pepsin cleaves the gamma chains on the C-terminal side of the inter-heavy chain disulfide bridges, resulting in one bivalent antigen-binding fragment F(ab’)2.

Skeletal Framework of IgG:

Skeletal Framework of IgG

3-D view of Lighter shade antigen area:

3-D view of Lighter shade antigen area

How are monoclonal antibodies formed?:

How are monoclonal antibodies formed?

Monoclonal Antibodies:

Monoclonal Antibodies

How are polyclonal antibodies formed?:

How are polyclonal antibodies formed?

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Polyclonal antibodies

Handling of Antibodies :

Handling of Antibodies In order to achieve optimal performance from the reagents used in immunohistochemistry, it is imperative to observe certain basic rules for their handling and storage. Properly maintained, most reagents will remain stable for months and even years. The recommendations given by the manufacturer on specification sheets and on vial labels should always be followed.

Receiving of antibodies:

Receiving Although most commercially produced immunochemicals are guaranteed to be stable for up to several years, reasonable precautions taken by the user will help maintain this stability. Upon receipt, immunochemicals should be promptly stored according to the manufacturer’s recommendations. The reagents should be logged in by entering the manufacturer’s and in-house lot numbers,expiration date,date of receipt and invoice number. These entries provide valuable information for the user, especially if later reclamations become necessary. Receiving of antibodies

Storage of antibodies:

Storage of antibodies Storage Perhaps the two most important considerations when storing antibodies are the storage container and the temperature. Storage containers The preferred materials for storage containers of protein solutions should have negligible protein absorptivity. Polypropylene, polycarbonate or borosilicate glass are particularly recommended and are used widely.

Protein concentration:

Solutions containing very low concentrations of protein(i.e. less than 1-10mg/dl), should receive an addition of bulk protein. Generally 0.1% to 1.0% bovine albumin is used to reduce loss through polymerization and absorption onto the container. Containers made of clear and colorless materials are preferable as these will allow ready inspection of contents Protein concentration

Temperature for storage of antibodies:

Temperature for storage of antibodies TEMPERATURE Probably more than any other factor, proper storage temperature as recommended by the manufacturer should be observed.Refrigerators and freezers used for storage of immunochemicals should be monitored for accurate and consistent temperatures.Valuable, or large quantities of immunochemical reagents should be stored in equipment with temperature alarm and emergency back-up power systems .

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Most prediluted antibodies and conjugates should be stored at 4-8degree C because freezing and thawing is likely to have a deleterious effect on these proteins . This also applies to entire kits which contain mostly prediluted reagents and monoclonal antibodies. Concentrated protein solutions, such as diluted antisera and immunoglobulin fractions, should be stored frozen in aliquots at -20 degree C or below to prevent cycles of repeated freezing and thawing. Frozen protein solutions should be brought to room temperature slowly, and higher temperatures should be avoided.

Handling and use of antibodies:

Handling and use of antibodies Handling and Use : Proper reagent care can reduce problems stemming from contamination, heat, excessive light exposure or expired shelf life. Reagent contamination can be avoided by the use of clean pipette tips. Prompt return of reagents to proper storage conditions will prolong shelf life.

Elimination of lipids:

Elimination of lipids The appearance of immunochemical reagents, particularly undiluted antisera, is rarely indicative of their subsequent performance. Although beta-lipoproteins have a very strong hydrophobic property, neither lipemia nor liposis in antisera has been studied systematically for interferences with immunohistochemical staining. Where obvious lipemia is encountered in an antiserum and thought to be the cause of interference with successful staining, removal of the lipids by use of dextran sulfate and calcium, or by extraction with organic solvents is recommended . Alternatively, the addition of 2g Aerosil (Degussa, NY) to 100ml antiserum followed by incubation for 4hr at 37degree C has proven useful.

Avoid excessive hemolysis:

Avoid excessive hemolysis Mild to moderate hemolysis in serum(plasma) resulting from incorrect bleeding techniques , probably does not interfere with most immunohistochemical staining procedures, but excessive hemolysis should be avoided. If excessive hemolysis or lipemia is encountered, isolation of the immunoglobulin fraction from the antiserum or normal serum may be necessary. Such isolates usually appear colorless and clear. It is unlikely that hemolyzed antisera induce “endogenous” peroxidase activity in tissue sections.

Antisera:

Antisera All antisera or normal nonimmune sera contaminated with bacterial growth should be discarded . Their use in immunohistochemical procedures most likely will introduce artifacts and nonspecific staining. Familiarity with the nature of antibodies, their capabilities and limitations, will allow the worker to better utilize these reagents and to more efficiently solve problems, if they occur. The following sections will further contribute to the understanding of antibodies; they also provide detailed information about the ancillary reagents and procedures used in immunohistochemistry.

Labels:

Labels Enzyme labels Colloidal metal labels Fluorescent labels Radiolabels

Enzyme Labels:

Enzyme Labels Horseradish peroxidase Alkaline phosphatase Bacterial-derived beta-D-galactosidase

Several alternative chromogens for demonstration of peroxidase:

Several alternative chromogens for demonstration of peroxidase Chromogens End product colour 3-amino-9-ethylcarbazole Red 4-chloro-1-naphthol Blue Hanker-Yates raegent Dark blue Alpha-naphtol pyronin Red-purple

Properties of Fluorochrome:

Properties of Fluorochrome Fluorochrome Excitation (nm) Emission (nm) Color DAPI 365 420 Blue Fluorescein 495 525 Green Hoechts 33258 360 470 Blue R-phycocyanin 555, 618 634 Red B-phycoerythrin 545, 565 575 Orange, red R-phycoerythrin 480, 545, 565 578 Orange, red Rhodamine 552 570 Red Texas red 596 620 Red

Colloidal metal labels:

Colloidal metal labels Immunogold method

Several alternative chromogens for demonstration of others labeled enzyme :

Several alternative chromogens for demonstration of others labeled enzyme Enzymes Chromogens Alkaline phosphatase Naphthol-AS-BI-Phosphate/New Fuchsin (NABP/NF) intense red Bromochloroindolyl Phosphate/Nitro Blue Tetrazolium (BCIP/NBT) intense black-purple Bacterial-derived beta-D-galactosidase 5-bromo-4-chloro-3indolyl- ß -D-galactopyranoside (BCIG)  intense blue product

BASIC ENZYMOLOGY:

BASIC ENZYMOLOGY Immunoenzymatic staining methods utilize enzyme –substrate reactions to convert colorless chromogens into colored end products. Of the enzymes used in these applications, horse-radish peroxidase, calf intestine alkaline phosphatase, glucose oxidase from Aspergillus niger and beta-galactosidase will be considered in some detail here. The various chromogens and substrates that can be used will also be described in this section, together with suggested procedures for the preparation of some substrate solutions.

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ENZYMES Enzymes are proteinaceous catalysts peculiar to living matter. Hundreds have been obtained in purified and crystalline form. Their catalytic efficiency is extremely high – one mole of a pure enzyme may catalyze the transformation of as many as 10,000 to 1,000,000 moles of substrate per min. While some enzymes are highly specific for only one substrate, others can attack many related substrates. A very broad classification of enzymes would include hydrolytic enzymes(esterases, proteases), phosphorylases, oxidoreductive enzymes(dehydrogenases, oxidases, peroxidases), transferring enzymes, decarboxylases and others.

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Enzymatic activity is dependent on several variables, such as enzyme and substrate concentrations, pH, salt concentration of the buffer milieu, temperature and light. Many enzymes also possess non-proteinaceous chemical portions termed prosthetic groups . Typical prosthetic groups are the iron-protoporphyrin of peroxidase, and biotin of CO2 transferases. In addition, many enzymes require the presence of metal ions such as Mg++, Mn+++,Zn++, which function as electrophilic(electron-attracting agents).

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The general formula which describes the reactions of an enzyme with its substrate may be written as follows: 1. Enzyme (E) + Substrate (S) ES complex 2. ES E+Products(P) Thus, before formation of the product, a transparent enzyme-substrate complex is formed at the active site(Prosthetic group) of the enzyme.

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Substances which interfere with the specific binding of the substrate to the prosthetic group are specific inhibitors and differ significantly from agents which cause nonspecific denaturation of enzyme (or any protein). Two basic types of inhibition are recognized – competitive inhibition and non-competitive inhibition . Competitive inhibition is the result of a reversible formation of an enzyme-inhibitor complex (EI). E+inhibitor(I)+S EI +S

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The formation of EI can be reversed by a change in the concentration of substrate and/or inhibitor, unless the affinity of I for E is greater than of S for E. The action of carbon monoxide or azides on the heavy metals of respiratory enzymes is a typical example of competitive inhibition. In noncompetitive inhibition, the inhibition depends solely on the concentration of the inhibitor and generally, is not reversible. Noncompetitive inhibition may or may not involve the prosthetic group of the enzyme and manifests itself by slowing down or halting the velocity of the enzymes reaction upon the substrate. E+I+S EIS

Selecting Enzymes:

Selecting Enzymes Selecting the enzyme most suitable for a particular immunohistochemical application depends on a number of criteria: The enzyme should be available in highly purified form and be relatively inexpensive. Conjugation (Covalent binding to antibody or avidin, for example) or noncovalent binding should not abolish enzyme activity, although it may diminish it. The bound enzyme should be stable in solution. endogenous enzyme activity should interfere only minimally with specific antigen-related staining. the products of the enzymic reactions should be readily detectable and stable.

Labels:

Labels Enzyme labels Colloidal metal labels Fluorescent labels Radiolabels

Horse Radish Peroxidase Label:

Horse Radish Peroxidase Label Horse Radish Peroxidase : This enzyme(molecular weight 40KD) is isolated from the root of the horseradish plant . Peroxidase has an iron-containing heme group (hematin) as its active site and in solution is colored brown . The hematin of peroxidase first forms a complex with hydrogen peroxide and then causes it to decompose resulting in water and atomic oxygen. Peroxidase oxidizes several substances, two of which are polyphenols and nitrates .

Inhibition of Peroxidase:

Inhibition of Peroxidase It should be noted that, similar to many other enzymes, peroxidase and some peroxidase-like activities can be inhibited by excess substrate. The complex formed between peroxidase and excess hydrogen peroxide is catalytically inactive and in the absence of an electron donor(eg. Chromogenic substance), is reversibly inhibited. It is the excess hydrogen peroxide and the absence of an electron donor that brings about quenching of endogenous peroxidase activities. Cyanide and azide are two other strong(reversible) inhibitors of peroxidase

Binding of Peroxidase:

Binding of Peroxidase Covalent Binding: Peroxidase can be attached to other proteins either covalently or noncovalently. The covalent binding of peroxidase to other proteins can be performed using either one-step or two-step procedures involving glutaraldehyde. The chemical 4,4’- difluoro-3,3’-dinitrophenyl sulfone(FNPS) is less commonly used for this purpose. In all cases, the epsilon-amino groups of lysine and N-terminal amino groups of both proteins are involved in this reaction.

Conjugation procedure:

Conjugation procedure The two-step conjugation procedure is preferred because, relative to the antibody molecule, the peroxidase molecule has a paucity of reactive groups. As a consequence, adding glutaraldehyde to a solution containing an admixture of peroxidase and antibody will result in more antibody molecules being conjugated to each other than to the enzyme. In the two-step procedure, peroxidase reacts with the bifunctional reagents first. In the second stage, only activated peroxidase is admixed with the antibody resulting in much more efficient labeling and no polymerization.

Conjugation with avidin:

Conjugation with avidin Peroxidase is also conjugated to avidin using the two-step glutaraldehyde procedure and is used in this form in the LAB procedure Conjugation with biotin also involves two steps as biotin must first be derivatized to the biotinyl-N-hydroxysuccinimide ester or to biotin hydrazide before it can be reacted with the epsilon-amino groups of the enzyme.

PAP complex:

NONCOVALENT BINDING: Noncovalent binding of peroxidase to antibody (also known as unlabelled antibody binding) is described in great detail by Sternberger . Instead of the use of bifunctional reagents, IgG –class antibodies to peroxidase are used to form a semicyclic soluble immune complex consisting of two antibody and three enzyme molecules. The molecular weight of the peroxidase-antiperoxidase or PAP complex is 400-430KD. PAP procedures have a much higher sensitivity than methods that utilize the antibody-peroxidase complex. PAP complex

CALF INTESTINE ALKALINE PHOSPHATASE:

CALF INTESTINE ALKALINE PHOSPHATASE Calf intenstine alkaline phosphatase (molecular weight of 100KD) removes (by hydrolysis) and transfers phosphate groups from organic esters by breaking the P-O bond; an intermediate enzyme-substrate(PO4) bond is briefly formed. The chief metal activators for alkaline phosphatase are Mg++,Mn++ and Ca++.

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Alkaline phosphatase had not been used extensively in Immunohistochemistry until publication of an unlabelled anti-alkaline phosphatase procedure(the APAAP procedure). The soluble immune complexes utilized in this procedure have molecular weights of approximately 560KD. The major advantage of the APAAP procedure compared to the PAP technique(which also uses soluble immune complexes) is the lack of interference posed by endogenous peroxidase activity. The APAAP technique is particularly recommended for use on blood and bone marrow smears . Endogenous alkaline phosphatase activity from bone, kidney, liver and some white cells is reportedly inhibited by the addition of 1mM Levamisole to the substrate solution, although 5mM has been found to be more effective. No other quenching step is required . Intestinal alkaline phosphatases are not adequately inhibited by levamisole.

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GLUCOSE OXIDASE(Aspergillus niger) This enzyme molecular weight 185KD has been found in several molds. Since its prosthetic group is composed of flavin, it is referred to as a flavoprotein(flavoenzyme). Flavin undergoes reduction by accepting hydrogen from the oxidation of glucose and passing it on to a receptor, such as oxygen:

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2. GOFH2+O2 Go-F+H2O2 There is no glucose oxidase in mammalian tissue, so no endogenous enzyme activity is encountered. Glucose oxidase has been utilized in labeled and unlabelled antibody techniques, as well as in the avidin-biotin technology. The chief disadvantage of glucose oxidase is its relatively low sensitivity when compared to peroxidase or alkaline phosphatase. Because of its low sensitivity, glucose oxidase requires the use of approximately 10-fold higher concentrations of primary antibodies.

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BETA-GALACTOSIDASE (Escherichia Coli) The beta-galactosidase isolated from E.Coli has a molecular weight of 500KD. When this enzyme is used at its optimal pH of 7.0 to 7.5, no interference from mammalian beta-galactosidase (Optimal pH of 5.5 to 6.0) will be encountered. Beta-galactosidase can be used in ELISA as well as in immunochemical procedures. When utilized in the latter, an insoluble indigo reaction product is obtained from the substrate 5-bromo-4-chloro-3-indoxyl-beta-D-galactoside.

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SUBSTRATES AND CHROMOGENS PEROXIDASE As described above, peroxidase activity in the presence of an electron donor first results in the formation of an enzyme-substrate complex, and then in the oxidation of the electron donor. The electron donor provides the “driving” force in continuing catalysis of H2O2, while its absence effectively stops the reaction. There are several electron donors which, upon being oxidized become colored products and are therefore called chromogens . This and the property of becoming insoluble upon oxidation, make such electron donors useful in Immunohistochemistry.

DAB:

DAB Three 3’-DIAMINOBENZIDINE TETRAHYDROCHLORIDE(DAB ) Produces a brown end product which is highly insoluble in alcohol and other organic solvents. Oxidation of DAB also causes polymerization , resulting in the ability to react with osmium tetroxide, and thus increasing its staining intensity and electron density . Of the several metals and methods used to intensify the optical density of polymerized DAB, gold chloride in combination with silver sulfide appears to be the most successful.

THREE-AMINO-9-ETHYLCARBAZOLE(AEC) Upon oxidation, AEC forms a rose-red end product which is alcohol soluble. Therefore, specimens processed with AEC must not be immersed in alcohol or alcoholic solutions(eg. Harris’ hematoxylin). Instead, an aqueous counterstain and mounting medium should be used. AEC is unfortunately susceptible to further oxidation and, when exposed to excessive light, will fade in intensity. Storage in the dark is therefore recommended. :

THREE-AMINO-9-ETHYLCARBAZOLE(AEC) Upon oxidation, AEC forms a rose-red end product which is alcohol soluble. Therefore, specimens processed with AEC must not be immersed in alcohol or alcoholic solutions(eg. Harris’ hematoxylin). Instead, an aqueous counterstain and mounting medium should be used. AEC is unfortunately susceptible to further oxidation and, when exposed to excessive light, will fade in intensity. Storage in the dark is therefore recommended .

Alkaline Phosphatase:

Alkaline Phosphatase ALKALINE PHOSPHATASE In the immunoalkaline phosphatase staining method, the enzyme hydrolyzes naphthol phosphate esters (substrate) to phenolic compounds and phosphates . The phenols couple to colorless diazonium salts (chromogen) to produce insoluble, colored azo dyes. Several different combinations of substrates and chromogens have been used successfully.

Naphthol AS-MX Phosphate, Fast Red TR and Fast Blue BB:

Naphthol AS-MX Phosphate, Fast Red TR and Fast Blue BB NAPHTHOL AS-MX PHOSPHATE, FAST RED TR AND FAST BLUE BB Naphthol AS-MX phosphate can be used in its acid form or as the sodium salt. The chromogens Fast Red TR and Fast Blue BB produce a bright red or blue end product respectively. Both are soluble in alcoholic and other organic solvents, so aqueous mounting media must be used. Fast Red TR is preferred when staining cell smears.

New Fuchsin:

New Fuchsin New Fuchsin also gives a red end product. Unlike Fast Red TR and Fast Blue BB, the color produced by New Fuchsin is insoluble in alcohol and other organic solvents, allowing for the specimens to be dehydrated before coverslipping. The staining intensity obtained by use of New Fuchsin is greater than that obtained with Fast Red TR and Fast Blue BB.

Four-chloro 1-Napthol:

Four-chloro 1-Napthol FOUR-CHLORO-1-NAPHTHOL(CN) Precipitates as a blue end product . Because CN is soluble in alcohol and other organic solvents, the specimen must not be dehydrated, exposed to alcoholic counterstains, or coverslipped with mounting media containing organic solvents. Unlike DAB, CN tends to diffuse from the site of precipitation.

Hanker Yates Reagent:

Hanker Yates Reagent p-PHENYLENEDIAMINE DIHYDROCHLORIDE/pyrocatechol (Hanker-Yates Reagent) Gives a blue-black reaction product which is insoluble in alcohol and other organic solvents. Like polymerized DAB, this reaction product can be osmicated. Varying results have been achieved with Hanker-Yates reagent in immunoperoxidase techniques.

OTHER SUBSTRATES AND CHROMOGENS: :

OTHER SUBSTRATES AND CHROMOGENS: Additional substrates include Naphthol AS-Bi phosphate, napthol As-TR phosphate and 5-bromo-4-chloro-3-indoxyl phosphate. Other possible chromogens include Fast Red LB, Fast Garnet GBC and Nitro Blue Tetrazolium (NBT).

GLUCOSE OXIDASE :

GLUCOSE OXIDASE There are several tetrazolium salts whose formazan reaction products are suitable for immuno-enzymatic staining methods with a glucose oxidase label. Previously, thiazoyl blue (MTT) has been used, but the colored end product is unstable even after chelation with a heavy end product is unstable even after chelation with a heavy metal. However, the chromogens INT, NBT, TNBT yield stable permanent preparations. Among recommended counterstains for these chromogens are Nuclear Fast Red, Metanil Yellow, Fast Green, Phloxine, aqueous acid fuchsin and methyl green . These can be used individually or in combination.

Glucose and INT:

Glucose and INT GLUCOSE AND INT Two-(p-iodophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride(INT) produces a red end product soluble in alcohol and organic solvents . Slides must be mounted from water with an aqueous mounting medium.

Nitra Blue Tetrazolium:

Nitra Blue Tetrazolium GLUCOSE AND NITRO BLUE TETRAZOLIUM Two,2’-di-p-nitrophenyl-5,5’-diphenyl-3,3’(3,3’-dimethozy-4,4’-diphenylene)-ditetrazolium chloride(NBT) produces a blue to black end product, slightly soluble in alcohol and other organic solvents . Therefore, it is recommended that sections stained with NBT be quickly dehydrated and cleared before coverslipping. NBT has been used successfully in double staining methods.

Tetranitro blue tetrazolium:

Tetranitro blue tetrazolium GLUCOSE AND TETRANITRO BLUE TETRAZOLIUM Two 2’,5,5’-tetra-p-nitrophenyl-3,3’-(3,3’-dimethoxy-4,4’-diphenylene) ditetrazolium chloride(TNBT) produces a brown-end product that is completely insoluble in alcohol and other organic solvents. Therefore, preparations stained with TNBT can be dehydrated and cleared before coverslipping

Basic immunochemistry :

Basic immunochemistry Antibody titer dilutions, as well as incubation time and temperature, are tightly interwoven in their effect on the quality of immunohistochemical staining. These factors can be changed independently or, as is more often the case, in complementary fashion, to bring about marked differences in the quality of staining. Generally, when making any changes, the overriding goal should be the achievement of optimal specific staining accompanied by minimal interference from background staining.

Antibody Titer:

Antibody Titer Antibody Titer In immunohistochemistry, an antibody titer is defined as the highest dilution of an antiserum which results in optimal specific staining with the least amount of background . This highest dilution is determined primarily by the absolute amount of specific antibodies present. In polyclonal antisera, antibody levels are generally expressed as micrograms of antigen precipitated per milliliter of antiserum. This can be of interest to the immunohsitochemist, but is not necessary information. For monoclonal antibody preparations, however, the absolute concentration of specific antibodies can be readily measured and frequently forms the basis for making the required dilutions.

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The highest dilution is also governed by the affinity of an antibody. If the titer is held constant, a high affinity antibody is likely to react faster with tissue antigen and give more intense staining in a short incubation period than an antibody of low affinity. The augmentation of titers by the isolation of immunoglobulin fractions from polyclonal antisera and their subsequent enrichment usually produces little benefit in immunohistochemical applications because nonspecific antibodies- frequently an additional source for nonspecific background- become enriched also.

Definition of Antibody titers:

Definition of Antibody titers Typically, antibody titers, as defined above, may vary from 1:5 to 1:100 for monoclonal antibodies in cell culture supernatants from 1:100 to 1:2000 for polyclonal antisera, and up to 1:1,000,000 and over for monoclonal antibodies in ascites fluid.

Antibody Dilution:

Antibody Dilution Antibody dilution Usually the manufacturer offers prediluted reagents ready for use, or recommends dilution ranges to be made compatible with other variables such as method, incubation time and temperature. If this information is not provided, optimal working dilutions of immunochemical reagents must be determined by titration. Correct dilutions will contribute to the quality of staining if they are prepared accurately and consistently. They are best determined by first selecting a fixed incubation time and then by making small volumes of a series of experimental dilutions. Depending on specimen size, applications of 0.2-0.4ml of solution per section is generally adequate .

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It should be noted that, atleast on paraffin sections, optimal dilutions of primary antibodies are not so frequently signaled by a peak in staining intensity alone, but rather by strong specific staining in the presence of minimal background (maximal signal-to-noise ratios). Probably because formalin fixation and paraffin embedding destroy most antigenic determinants, use of even excessively high primary antibody concentrations rarely causes an inhibition in the formation of immune complexes or prozone-like effects. Once the optimal working dilution has been found, larger volumes can be prepared according to need and stability.

Preparation of dilutions:

Preparation of dilutions Dilutions are usually expressed as the ratio of the more concentrated stock solution to the total volume of the desired dilution. For example, a 1:10 dilution is made by mixing one part of the stock solution with nine parts of diluent giving a total of ten parts. Two-fold serial dilutions are made by successive 1:2 dilutions of the previous stock dilution. In order to make a very small volume of a highly diluted solution, it may be necessary to make it in two steps. For example, to prepare 1.0ml of a 1:1000 dilution, first make 100microliter of a 1:10dilution(10microliter+90microliter), and then 1000microliter of a 1:100 dilution using 10microliter of the intermediate dilution(10microliter+990microliter). The more concentrated dilution (1:10)can be stored frozen.

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When making dilutions, the use of adjustable pipets allows for greater flexibility and more accurate delivery . To measure volumes in excess of 1.0ml, serological or volumetric pipets can be used. This indicates the volumes of stock reagents and diluents to be mixed to obtain various dilutions ranging from 1:5 to 1:2000. Checkerboard titrations are used to determine the optimal dilution of more than one reagent simultaneously . In the following checkerboard titration, the optimal dilutions of a primary antibody and the PAP reagent are to be found, while the dilution of the link antibody is held constant(not shown). Nine tissue sections are required if three dilutions are to be tested.

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PAP Complex Primary antibody Dilutions 1:50 1:50 1:100 1:200 1:100 1:50 1:100 1:200 1:200 1:50 1:100 1:200 As noted earlier, staining results achieved by use of several different dilutions will often be identical or similar, in which case the cost of the reagent may become a factor in selecting the optimal dilutions.

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It should be noted that precise definition of the optimal signal-to-noise ratio as a function of the primary antibody dilution was found to be more critical with immunohistochemical methods using unlabelled enzyme-antienzyme complexes (PAP,APAAP), than with methods utilizing the avidin-biotin technology. This is probably consistent with the observation that, as opposed to the PAP method, the ABC method cannot distinguish between high and low concentrations of tissue antigens.

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Antibody Incubation Incubation time, temperature,antibody titer are interdependent; a change in one factor will affect the others. Incubation Time There is an inverse relationship between incubation time and antibody titer- the higher the antibody titer, the shorter the incubation time can be. In practice, however, it is expedient to first set a suitable incubation time before determining the optimal antibody dilution. Conversely, higher concentrations of specific antibodies (and higher affinities) allow for the shortening of the incubation time.

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Incubation times for the primary antibody may vary from 1.5 min to 48hr, with 20-30min probably being the most widely used. For an antibody to react sufficiently strongly with the bound antigen in 1.5min, it must be of high affinity and relatively high concentration. Variables believed to contribute to increased nonspecific background staining should be kept to a minimum. Primary antibody incubations of 48hr duration allow, more than anything else, for great economy because very high dilutions of antiserum may be used. While antisera of low affinity and/or low titer must be incubated for long periods in order to reach equilibrium, nothing can be gained by prolonging primary antibody incubation beyond the time at which the tissue antigen is saturated with antibody. Because equilibrium is usually not reached during primary antibody incubations of less than 20min, consistent timing of this step is of great importance. Inconsistent incubation times can cause variations in the intensity and overall quality of staining will require the use of a humidity chamber.

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Incubation Temperature Because equilibrium in antigen-antibody reactions is reached more quickly at 37degree C as compared to room temperature, some workers prefer to incubate at the higher temperature. An increase in incubation temperature allows for a greater dilution of the antibody; if the dilution is not increased, the incubation time may be shortened . It is not known whether temperature selectively promotes the antigen-antibody reaction rather than the reaction that gives rise to background. A temperature of 4degreeC is frequently used in combination with overnight or longer incubations. Slides incubated for extended periods or at 37degreeC should be placed in a humidity chamber to prevent evaporation and drying of the tissue sections. Similarly, tissue incubated at room temperature in a very dry or drafty environment

The methods of Identifying Tissue Antigens:

The methods of Identifying Tissue Antigens Direct labeling of antibody Secondary antibody labeling

Direct Method :

Direct Method Tissue Antigen Labeled Antibody

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Immunocytochemical methods Traditional Direct technique EPOS staining method

EPOS Technique:

EPOS Technique

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Indirect technique New indirect technique(Dextran Polymer conjugate two-step visualization system) DAKO Catalyzed signal amplification(CSA) system Unlabelled antibody enzyme-complex techniques(PAP and APAAP) Immunogold Silver staining Technique(IGSS) Avidin-Biotin technique Hapten-labelling technique Mirror Image Complementary antibody labelling technique (MICA)

Two-Step Indirect Method :

Two-Step Indirect Method Tissue Antigen Primary Antibody Secondary Antibody

Dextran Conjugated Polymer Two step Visualization system:

Dextran Conjugated Polymer Two step Visualization system

ABC Method (avidin-biotin complex method ) :

ABC Method (avidin-biotin complex method )

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Conventional ABC technique

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Enzyme and chromogen substrate added

PAP Method (peroxidase anti-peroxidase method) :

PAP Method (peroxidase anti-peroxidase method)

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IGSS technique

SP Method (streptavidin peroxidase conjugated method):

SP Method (streptavidin peroxidase conjugated method )

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APAAP technique

Multiple labelling technique:

Multiple labelling technique

Steps of Immunohistochemistry:

Steps of Immunohistochemistry Tissue sections are incubated with primary antibodies against the target antigen. The specimens are then washed and a secondary antibody against primary antibody is added. The secondary antibodies are often biotinylated, that is, conjugated to biotin, a vitamin with an extremely high affinity for the protein streptavidine.

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A complex made of streptavidin and the enzyme peroxidase is used for staining. The addition of a chromogenic substrate such as diaminobenzidine (DAB) or aminoethylcarbazole (AEC), leads to a color staining reaction that accurately reflects the distribution of the target antigen in the tissue sample.

Unmasking of Antigen Sites:

Unmasking of Antigen Sites Tissue fixation on routinely histotechnique can masked antigens because of formalin cross-linking or even destroys some antigenic epitopes . The retrieval techniques of unmasked antigens : (1) proteolytic enzyme digestion (2) microwave (3) microwave and trypsin (4) pressure cooker

Contents:

Contents Incubation methods (Manual/Automated) Fixation and paraffin-wax block immunocytochemistry Antigen retrieval techniques Proteolytic enzyme methods Trypsin method Protease method Pepsin method Heat mediated antigen retrieval Microwave oven heating Coplin jar method Recommendation on the use of expensive Commercial retrieval fluids Pressure cooker antigen retrieval Combination of microwave oven heating and trypsin or chymotrypsin digestion

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Microwave Oven

Labels, Background Staining, DAB Solutions:

Labels, Background Staining, DAB Solutions

Buffer Solutions:

Buffer Solutions

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Pressure Cooker

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Multiple chamber couplin Jar

Coplin Jar:

Coplin Jar

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Steamer

Antigen Recognition:

Antigen Recognition 1’Ab : primary antibody 2’Ab : secondary antibody B : biotin SA : streptavidine HRP : horseradish peroxidase Target is antigen (protein argets) Blocking reagent : BSA (bovine serum albumin ) The sandwich of antigen recognition on tissue samples.

Contents:

Contents Enhancement and amplification Increasing the concentration of primary antibody. Prolonging incubation Increasing the concentration of bridge reagent Chemical enhancement of the reaction end-product of the peroxidase-diamiobenzidine(DAB) method Repeated applications of the bridge and label Changing the chromogen substrate used Techniques for elevating the sensitivity of the EPOS method and other pre-diluted reagents Tyramine signal amplification system Multiple labels

IHC STAINING TRAYS:

IHC STAINING TRAYS

FROZEN SECTION IN CRYOSTAT:

FROZEN SECTION IN CRYOSTAT

MICROTOME :

MICROTOME

EMBEDDING PROCEDURE:

EMBEDDING PROCEDURE

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Slide container

MOUNTING:

MOUNTING

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Purpose of blocking endogenous enzymes

FITC signal transduction system:

FITC signal transduction system

Contents:

Contents Immunohistochemistry in practice Workload Choice of technique for preservation of antigens( Frozen sections and cytological preparations) Formalin – Fixed, routinely processed paraffin-wax sections Blocking endogenous enzymes Blocking background staining Controls Practical aspects of immunocytochemical staining Dilution of immune serum/antibodies Washes

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Reagent and procedure controls are necessary for the validation of immunohistochemical staining results. Without their use, interpretation of staining, in itself a subjective art, would be haphazard, and the results would be of doubtful value.

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REAGENT CONTROLS Control of reagents is best performed by both manufacturer and user within optimized quality control programs. The aim is to ascertain whether the primary and secondary antibodies are specific for their target antigens. To this end, it is best to test the primary antibody first for optimal dilution on tissue and then against an expanded panel of tissues known to either contain or not contain the tissue marker.

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Various immunochemical techniques,such as double diffusion, immunoelectrophoresis and rocket electrophoresis may be useful for obtaining additional information. The secondary (link) antibody should be affinity-absorbed in order to render it nonresective to human tissue proteins(immunoglobulins). Quality control programs should be supplemented by proper record keeping on dilutions, diluents, incubation times, and dates on which any procedural changes are introduced.

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PROCEDURE CONTROLS Procedure Controls serve to ascertain primarily whether the staining protocols were followed correctly, whether day-to-day and worker-to-worker variations have occurred, and whether reagents continue to be in good working order. Procedure controls involve reagent substitution and tissue controls. REAGENT SUBSTITUTION Of all the components used in an immunochemical staining system, the primary antibody is, without doubt, the most critical, although occasionally other reagents may need to be replaced.

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Primary Antibody To ascertain its specificity in Immunohistochemistry on a day-to-day basis, the primary antibody should be replaced with either the affinity-absorbed antiserum, with another irrelevant antibody, or with preimmune or non-immune serum from the same species that produced the primary antibody. Substitution of diluent for the primary antibody or complete omission of the primary antibody step is not recommended. Affinity absorption of the primary antibody with highly purified antigen is the ideal means to obtain a valid negative control for differentiating specific from non-specific staining.

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The problem is that purified antigen is rarely available in clinical histology laboratories, and that such preparations are very expensive. Practically, therefore most laboratories use nonimmune serum from the same species or antisera of irrelevant specificity. When the primary antibody is a monoclonal, use of another irrelevant antibody is probably the best negative reagent control, although tissue culture medium used to propagate monoclonal antibodies is frequently substituted. The paramount objective in the selection of a good control in all cases is to imitate all facets of the primary antibody except for the antigen specificity.

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Not only should whole antiserum or its IgG fraction site be replaced by normal serum or nonimmune IgG, respectively, but the IgG fractions should contain nearly identical protein concentrations. It is recommended that antibody solutions and controls be of comparable age(+_1Yr) and, hence, likely to contain similar amounts of protein aggregates. The aggregates may contribute to nonspecific staining. Buffers used for the dilution of antibodies and controls must be identical. If these points are not observed, much confusion may result from background staining in the positively stained section, but not in the negatively processed control, or vice versa.

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A method for the preparation of a negative reagent control is illustrated below:- Example:- Primary Antibody: An immunoglobulin fraction of rabbit antiserum. Protien concentration : 4.8g/l Recommended Dilution : 1:200

Calculation of Required dilution:

Calculation of Required dilution Appropriate negative reagent control:Nonimmune rabbit immunoglobulin fraction Protien concentration : 20g/l Required dilution : To be determined. Step 1: Calculation of protein concentration of diluted primary antibody. 4.8g/l ----------------- = 0.024g/l 200 Step 2: Calculation of dilution of nonimmune rabbit immunoglobulin fraction to give protein concentration equivalent to that of diluted primary antibody. 20g/l ------------ = 833 0.024g/l Dilution = 1: 833 (One part of non immune rabbit immunoglobulin fraction in 800parts buffer would be acceptable).

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OTHER REAGENTS After the primary antibody has been ruled out as the causative agent, other reagents may need to be replaced. Substitution of link(labeled) antibody with nonimmune serum from the same species, or of old PAP(APAAP) reagent with different lots, may help in explaining the origin of nonspecific staining. TISSUE CONTROLS Tissue controls can be either the negative, positive or internal type. NEGATIVE TISSUE CONTROLS These specimens are processed(fixed, embedded) identically to the unknown, but donot contain the relevant tissue marker. One example, would be normal liver compared to hepatitis B surface antigen-positive liver.

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POSITIVE TISSUE CONTROLS These controls also are processed identically to the specimen, but they must contain the target protein. In some cases, it will be advantageous to have this control tissue stain marginally positive, so as to monitor not only for the presence of the antigen, but also any possible loss of sensitivity. This loss might not be apparent if only intensely staining controls are used. Controls for loss of sensitivity would be particularly important when staining tumors, for example, in this case, staining intensity frequently varies with the degree of tumor differentiation.

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INTERNAL TISSUE CONTROLS This type of control, also known as “built-in control, is ideal because the variables of tissue preparation, processing and staining are eliminated. Built-in controls contain the target marker in the tumor to be identified, and in adjacent normal elements. On example is the presence of S-100 protein in melanoma and normal tissue elements, such as peripheral nerves and melanocytes. Built-in controls have the additional advantage that no separate positive control sections are required.

Signal Amplification:

Signal Amplification

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Enzyme and chromogen substrate added

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LIST OF ORGANS FOR HISTOPATHOLOGICAL ANALYSIS: Neural Respiratory: Brain : Cerebrum, Lungs and trachea Olfactory, Cerebellum Other: Spinal cord and peripheral nerves Eyes, Inner ear, nasal passages Vascular: Hematologic: Heart and blood vessels Spleen, Thymus, Bone Marrow Lymph nodes and Peyer’s patches Integument: GastroIntestinal: Skin, Bone, Cartilage Liver, Salivary Gland, Pancreas Skeletal muscle, Stomach and Duodenum, Stroma and Adipose tissue Small intestine (Ileum) Large intestine (Colon), Cecum GenitoUrinary Endocrine : Kidney, Bladder Adrenals, Pituitary Uterus, Ovary, Fallopian tubes Thyroid , Parathyroid Testis, Prostate, Breast, Placenta

General Immunohistochemistry Protocol:

General Immunohistochemistry Protocol

Applications:

Application s Cancer diagnostics differential diagnosis Treatment of cancer Research

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STAINING METHODS There are many immunoenzymatic staining methods which can be used to localize antigens. The choice is based on the individual needs of each laboratory, such as the type of specimen being investigated, the degree of sensitivity required, and the processing time and cost requirements. This section describes the most commonly used immunoenzymatic staining methods for light microscopy. Brief examples of working procedures will be presented and the advantages and disadvantages of each will be discussed. There are a couple of general considerations regarding buffers that the user should keep in mind. Sodium azide, an antibacterial agent present in many commercially-prepared buffers, can prevent binding of the peroxidase enzyme to its substrate and inhibit color development. Thus, the use of sodium azide in wash and diluent buffers is not recommended.

Buffer:

Buffer Also, phosphate buffer should not be used in procedures involving alkaline phosphatase, as activity of the enzyme will be inhibited. (The substitution of distilled water for wash buffer has been tested in several staining systems and found to have no adverse effect on the staining results). The following procedures are guidelines only; the user should decide upon the optimal format and appropriate label.

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Direct method In this technique, an enzyme-labelled primary antibody reacts with the antigen in the tissue. Subsequent use of substrate and chromogens concludes the reaction sequence. Because this method utilizes only one antibody, it can be completed quickly, and non-specific reactions are limited. However, since staining involves only one labeled antibody, little signal amplification is achieved. This method is now used only rarely. General Procedure : All incubations are carried out at room temperature. Gently, rinse slide with distilled water or buffer from a wash bottle. Place slide in buffer bath for 5 minutes.

Steps of preparation:

Steps of preparation Remove excess liquid from around the specimen. Apply 4-6 drops of normal serum, diluted 1:5-1:20. Incubate 20-30min. Tap off serum and wipe away excess. Do not rinse. Apply 4-6 drops of enzyme-conjugated primary antibody, diluted approximately. Incubate 20-30min. Repeat steps 1 and 2. Apply substrate-chromogen solution and incubate until desired color intensity has developed. Rinse gently with distilled water from wash bottle. Counter-stain and coverslip.

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TWO- STEP INDIRECT METHOD In this method an unconjugated primary antibody binds to the antigen. An enzyme-labelled secondary antibody directed against the primary antibody(now the antigen) is then applied, followed by the substrate-chromogen solution. If the primary antibody is made in rabbit or mouse, the secondary antibody must be directed against rabbit or mouse immunoglobulins, respectively.

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This method is more versatile than the direct method because a variety of primary antibodies from the same species can be used with the same labeled secondary antibody. The procedure is also several times more sensitive than the direct method because several secondary antibodies are likely to react with different epitopes on the primary antibody. As a consequence, this attaches more enzyme molecules at the site of the antigen, and hence results in greater sensitivity. Undesired reactions may occur if the secondary antibody cross-reacts with endogenous immunoglobulins in the specimen. This cross-reactivity, however can be eliminated by using preabsorbed antiserum, that is secondary serum which has been absorbed with immunoglobulin from the species from which the specimen is obtained.

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Probably, one of the most frequent applications of the indirect technique is the detection of autoimmune antibodies in human serum. In this case, the patient’s serum is the primary antibody and is applied to a tissue specimen containing the antigen under study. An enzyme-linked secondary antibody to human immunoglobulin is then added. If the serum contains antibodies to the antigen, the enzyme-linked secondary antibody will bind to the patient’s antibody and thereby localize the antigen.

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GENERAL PROCEDURE FOR USE WITH POLYCLONAL RABBIT(OR MONOCLONAL MOUSE) PRIMARY ANTIBODY All incubations are carried out at room temperature. Gently rinse slide with distilled water or buffer from a wash bottle. Place slide in buffer bath for 5min. Remove excess liquid from around the specimen. Apply 4-6drops of normal swine(rabbit) serum, diluted 1:5 – 1:20. Incubate 20-30min. Tap off serum and wipe away excess. DO NOT RINSE. Apply 4-6drops of rabbit(mouse) primary antibody, diluted appropriately. Incubate 20-30min. Repeat steps 1 and 2. Apply 4-6drops of enzyme-conjugated swine(rabbit) antibody directed against rabbit (mouse) immunoglobulins, diluted appropriately. Incubate 20-30min.

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Repeat steps 1 and 2. Apply substrate-chromogen solution and incubate until desired color intensity has developed. Rinse gently with distilled water from wash bottle. Counter-stain and coverslip.

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THREE-STEP INDIRECT METHOD In the three-step indirect method, a second enzyme-conjugated antibody layer is added to the previously described indirect technique. The primary and enzyme-conjugated secondary antibody are applied sequentially, followed by a third enzyme-conjugated antibody specific to the secondary antibody. For example, if the secondary antibody was made in goat, the third antibody must be specific for goat immunoglobulin. Both secondary and tertiary antibodies must be conjugated to the same enzyme. The addition of a third layer of antibody serves to further amplify the signal, since more antibodies are capable of binding to the previously bound secondary reagent. This places additional enzyme at the site of the tissue antigen and thereby produces greater color intensity. The enhanced signal is particularly helpful when staining antigens with a limited number of epitopes. The three-step indirect method provides a simple way to increase staining intensity. The sensitivity of this technique provides a good alternative to soluble immune complex and avidin-biotin procedures.

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GENERAL PROCEDURE FOR USE WITH MONOCLONAL MOUSE PRIMARY ANTIBODY All incubations are carried out at room temperature. Gently rinse slide with distilled water or buffer from a wash bottle. Place slide in buffer bath for 5min. Remove excess liquid from around the specimen. Apply 4-6 drops of normal swine serum, diluted 1:5-1:20. Incubate 20-30min. Tap off serum and wipe away excess. DO NOT RINSE. Apply 4-6 drops of monoclonal mouse primary antibody, diluted appropriately. Incubate 20-30min. Repeat steps 1 and 2. Apply 4-6 drops of peroxidase-conjugated rabbit antibody directed against mouse immunoglobulins, diluted appropriately. Incubate 20-30min. Repeat steps 1 and 2. Apply 4-6drops of enzyme-conjugated swine antibody directed against rabbit immunoglobulins, diluted appropriately. Incubate 20-30min. Repeat steps 1 and 2. Apply substrate chromogens solution and incubate until desired color intensity has developed. Rinse gently with distilled water from wash bottle. Counterstain and coverslip.

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SOLUBLE ENZYME IMMUNE COMPLEX METHOD This method, sometimes also called the unlabelled antibody method, utilizes a preformed soluble enzyme-antienzyme immune complex. This type of immune complex consists of an enzyme(the antigen), and the antibody directed against the enzyme. To obtain a soluble enzyme-antienzyme complex, the enzyme is added in excess and any precipitate is removed. The staining sequence is as follows: unconjugated primary antibody, secondary antibody, soluble enzyme-antienzyme complex, substrate solution..

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The primary antibody and the antibody of the enzyme immune complex must be made in the same species so that the secondary antibody can link the two together. The secondary antibody, also called link antibody, has to meet two requirements: first, it must be directed against immunoglobulins of the species producing the primary antibody and enzyme immune complex; second it must be added in excess so that one of its Fab sites binds to the primary antibody from the enzyme immune complex

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Soluble enzyme-antienzyme immune complex techniques are named after the particular enzyme immune complex they use. For example, the PAP method utilizes a peroxidase-antiperoxidase complex, APAAP uses an alakaline phosphatase-antialkaline phosphatase complex, GAG uses glucose oxidase-antigluclose oxidase and so forth. The most commonly used methods are PAP and APAAP. The PAP complex-consists of three molecules of peroxidase and two antibodies against peroxidase enzyme. The APAAP complex has two molecules of alkaline phosphatase and one antibody against the enzyme.

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Soluble enzyme immune complex methods are among the most sensitive immunochemical techniques. The technique makes use of the natural affinity of antibody for antigen by using a stable immune complex as opposed to the harsher chemical conjugation process. The considerably greater degree of sensitivity compared to the previously described methods is mainly attributable to more enzyme molecules being localized per antigenic site. The enzyme-antienzyme complex method gives excellent results on fixed, paraffin embedded specimens. The PAP method is a commonly used technique for demonstrating tissue and cell antigens by light microscopy. The APAAP method allows the staining of specimens rich in endogenous peroxidase. It is particularly useful for blood smears where quenching with hydrogen peroxide frequently denatures leucocyte antigens.

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GENERAL PAP PROCEDURE FOR USE WITH POLY-CLONAL RABBIT(OR MONOCLONAL MOUSE) PRIMARY ANTIBODY All incubations are carried out at room temperature. Gently rinse slide with distilled water or buffer from a wash bottle. Place slide in buffer bath for 5 min. Remove excess liquid from around the specimen. Apply 4-6 drops of normal swine(Rabbit) serum, diluted 1:5-1:20. Incubate 20-30min. Tap off serum and wipe away excess. DO NOT RINSE. Apply 4-6 drops of rabbit(mouse) primary antibody, diluted appropriately. Incubate 20-30min. Repeat steps 1 and 2. Apply 4-6 drops of swine anti-rabbit(rabbit anti-mouse) antibody, diluted appropriately. Incubate 20-30min. Repeat steps 1 and 2. Apply 4-6 drops of horseradish peroxidase-rabbit(mouse) antiperoxidase(PAP) complex, diluted appropriately. Incubate 20-30min. Repeat steps 1 and 2. Apply peroxidase substrate-chromogen solution to give colored end product and incubate until desired color intensity has developed. Rinse gently with distilled water from wash bottle. Counterstain and coverslip.

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GENERAL APAAP PROCEDURE FOR USE WITH MONO-CLONAL MOUSE PRIMARY ANTIBODY In this procedure, the intensity of the reaction may be increased by repeating the incubation steps with the link antibody and APAAP complex. A separate step for quenching endogenous alkaline phosphatase activity is not necessary, as the quenching agent can be incorporated in the substrate reagent. A separate blocking step is not necessary since the link antibody is diluted in normal serum. All incubations are carried out at room temperature. 1.Apply 4-6 drops of supernatant mouse monoclonal primary antibody, diluted appropriately, incubate 30min. 2. Gently rinse slide with buffer from a wash bottle. Place slide in buffer bath for 1min.

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3. Remove excess liquid from around the specimen. 4. Apply 4-6drops of rabbit anti-mouse antibody, diluted appropriately with buffer containing normal serum. Incubate 30min. 5. Repeat steps 2 and 3. 6. Apply 4-6 drops of alkaline phosphatase-mouse antialkaline phosphatase(APAAP) complex, diluted appropriately. Incubate 30min. 7. Repeat steps 2 and 3. 8. Again apply 4-6 drops of rabbit anti-mouse antibody, diluted as in step 4. Incubate 10min. 9. Repeat steps 2 and 3. 10. Again apply 4-6 drops mouse APAAP complex, diluted as in step 6. Incubate 10min.

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11. Repeat steps 2 and 3. 12. Apply alkaline phosphatase substrate-chromogen solution to give colored end product and incubate until desired color intensity has developed. 13. Rinse gently with distilled water from wash bottle. Counterstain and coverslip. The repeated application of the link antibody and APAAP complex are optional steps which place additional enzyme at the antigenic site, resulting in a more intense color reaction. The signal enhancement achieved by repetition of antibody incubations is not confined to the APAAP method; it can be used with other procedures when greater staining intensity is desired.

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AVIDIN – BIOTIN METHODS These methods utilize the high affinity of avidin or streptavidin for biotin(dissociation constant 10-19 M). Avidin has four binding sites for biotin. However, due to the molecular orientation of the biotin binding sites, fewer than four molecules of biotin will actually bind. Currently, two avidin-biotin methods are in frequent use-the avidin biotin(LAB) technique. Both methods require a biotinylated antibody as a link antibody. Biotinylation is a mild process, whereby biotin is covalently attached to the antibody. Open sites on avidin from the avidin-biotin complex or enzyme-labelled avidin bind to the biotin on the link antibody. The biotinylated antibody does not have to be added in excess since free Fab sites are not needed for binding.

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The sequence of reagent application is primary antibody biotinylated secondary antibody, preformed avidin-biotin-enzyme compled(ABC) or enzyme-labelled avidin(LAB), substrate solution. Horsereadish peroxidase and alkaline phosphatase are commonly used enzyme labels. The strong affinity of avidin for biotin and the mild biotinylation process make the avidin-biotin methods more sensitive than the previously described direct and indirect methods. The original authors of the ABC method found the procedure to be greater in sensitivity than the PAP method. Subsequently, the LAB method was described to be approximately four- to eight- fold more sensitive than the ABC method.

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Much of the early work with the ABC method focused on the identification of pituitary hormones in normal and neoplastic tissue. Today, both ABD and LAB methods lend themselves to the localization of numerous antigens in a variety of specimens. As with the immune complex methods, excellent results can be achieved on fixed paraffin-embedded specimens. Some tissues such as liver and kidney contain endogenous biotin (EABA) which must be blocked to avoid nonspecific staining

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GENERAL ABC PROCEDURE FOR USE WITH POLY-CLONAL RABBIT(OR MONOCLONAL MOUSE) PRIMARY ANTIBODY To form the ABC complex, the solutions of the Avidin and biotinylated enzyme must be added in an optimal ratio and be prepared at least 30 min before use. All incubations are carried out at room temperature. Gently rinse slide with distilled water or buffer from a wash bottle. Place slide in buffer bath for 5 min. Remove excess liquid from around the section. Apply 4-6 drops of normal swine(rabbit) serum, diluted 1:5-1:20. Incubate 20-30min. Tap off serum and wipe away excess. Do not rinse. Apply 4-6 drops of rabbit(mouse) primary antibody, diluted appropriately.Incubate 20-30min.

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Repeat steps 1 and 2. Apply 4-6 drops of biotinylated swine anti-rabbit (rabbit anti-mouse) antibody, diluted appropriately. Incubate 20-30min. Repeat steps 1 and 2. Apply 4-6drops of avidin-biotin complex(mixed and diluted appropriately at least 30min before use.). Incubate 20-30min. Repeat steps 1 and 2. Apply substrate-chromogen solution and incubate until desired color intensity has developed. Rinse gently with distilled water from wash bottle.counter-stain and coverslip.

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GENERAL LAB PROCEDURE . When enzyme-labelled avidin is used, the same protocol should be followed as for the ABC procedure, except for Step 10, where appropriately diluted enzyme-labelled avidin is applied. It should be noted that the biotinylated antibody and the enzyme-labelled avidin can be pre-mixed and applied as a complex, thereby shortening this procedure by one step. QUICK STAINING Situations arise when rapid histopathological evaluation becomes either necessary or desirable. For example, during surgery. This need was first expressed almost 100years ago. Until recently, such evaluation was based almost entirely on morphological parameters following routine hematoxylin and eosin staining. The development of high-quality monoclonal and polyclonal antibodies, as well as the evolution of increasingly more sensitive staining techniques., has made possible to rapidly obtain immunochemical parameters to complement morphology.

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ANTIBODIES Experience has shown that one of the more important prerequisites for successful rapid immunohistochemical staining is the availability of high-quality primary antibodies. Antibodies of the highest affinity are required, and should be present in high titer. These features, combined with the application of the antiserum in lower dilutions than those traditionally used, result in successful rapid staining without interference by excessive background. Lower tittered antisera have a greater proportion of undesired antibodies which increases nonspecific background staining significantly. Because background is mostly the result of hydrophobic and ionic interactions between the proteins of the antiserum and the tissue, careful selection of the antiserum diluent will significantly contribute to a further reduction in background staining.

Background :

Background Unfortunately, measures that decrease the background caused by ionic interactions will often increase the background caused by hydrophobicity. Selection of buffer salt, pH, concentration and protein additives to the primary antibody diluent will therefore have to be made empirically and by studying the type of tissues on which the primary antibody will be used most frequently. Although background is less problematical with use of monoclonal antibodies, it has nevertheless been observed and occasionally the above countermeasures are required. In the more traditional and time-consuming procedures which employ higher antibody dilutions, longer incubation times, and repeatedly lengthy buffer baths, background staining is less of a problem

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STAINING METHOD The sensitivity of an immunohistochemical staining procedure depends to a large degree on antibody quality. When good antibody quality is combined with a highly sensitive detection technique the essential conditions for rapid staining have been fulfilled and need only be optimized(in accordance with principles described in Basic Immunohistochemistry ). . It is of vital importance,once reagents and techniques have been optimized, to follow the outline of the rapid staining procedure consistently and to use properly fixed tissue. Inconsistencies in the use of reagents and in the execution of methodological detail will almost always result in unacceptably scant specific staining or high non-specific background. Compared to the more traditional methods, quick staining requires that all reagents be used in higher concentration and for much shorter incubation periods; thus, relatively small errors in dilution or time will have a proportionately greater effect on the outcome of the quick staining.

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For this reason, all reagents should be ready for use before beginning the actual rapid staining procedure. No lengthy buffer baths are required routinely. However, during unavoidable interruptions, slides can be left in buffer baths. Similarly, lengthy incubations with various protein solutions for quenching nonspecific staining are not necessary, provided the tissue was prepared optimally and reagents and techniques and applied correctly.

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It is at times difficult to maintain consistency in the rapid staining protocol, particularly when a large number of slides must be processed simultaneously. For this reason, staining cassettes have been developed which can accommodate several glass slides. Within the cassette, these slides can be processed simultaneously and therefore identically, and with less manual effort than if handled individually. Staining can be accomplished without compromising the specific staining intensity and without any increase in undesired and without any increase in undesired background.

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It is possible that such devices may also offer greater economy in the use of sometimes expensive reagents. It is probably safe to predict that demand for rapid staining of tissue and cell specimens is likely to increase as long as sensitivity and specificity are not compromised. Within three to five years, tissue and cell staining will probably employ “dip-stick” methods similar to those used today in clinical chemistry testing.

Background :

Background Background staining is probably the most frequently encountered problem in Immunohistochemistry.

Hydrophobic interaction:

Hydrophobic interaction Hydrophobicity is a property shared, to varying degrees of most proteins. It is imparted to the peptided primarily by the side chains of the neutral aromatic amino acids phenylalanine, tyrosine and tryptophan. In addition, other amino acids, which have little attraction for water molecules, and to link to one another, further expelling water from the molecule. Hydrophobicity is one to the natural forces that confers stability on the tertiary structure of peptides. However, hydrophobic binding may also take place between different protein molecules, and thus impart stability to immune complexes. The hydrophobicity of soluble proteins may increase proteins storage due to aggregation, polymerization, or conjugation to other proteins.

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In tissue, proteins are rendered more hydrophobic by fixation with aldehyde-containing reagents such as formalin and glutaraldehyde. The increased hydrophobicity is often the result of cross-linking of reactive epsilon and alpha-amino acids, both within and between adjacent protein molecules. Because the extent of hydrophobic cross-linking of tissue proteins is primarily a function of tissue fixation, it follows that changes in fixation procedures will likely result in variable hydrophobicity. Factors such as time, temperature, pH during fixation should be optimized to avoid excessive cross-linking of tissue proteins. Tissues which aid to show background staining as a result of hydrophobic as well as the ionic) interactions, include connective tissue(Collagen, laminin, elastin, proteoglycans and others) epithelium(keratins?) and adipocytes(Lipids).

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The increased hydrophobicity is often the result of cross-linking of reactive epsilon and alpha-amino acids, both within and between adjacent protein molecules. Because the extent of hydrophobic cross-linking of tissue proteins is primarily a function of tissue fixation, it follows that changes in fixation procedures will likely result in variable hydrophobicity. Factors such as time, temperature, pH during fixation should be optimized to avoid excessive cross-linking of tissue proteins. Tissues which aid to show background staining as a result of hydrophobic as well as the ionic) interactions, include connective tissue(Collagen, laminin, elastin, proteoglycans and others) epithelium(keratins?) and adipocytes(Lipids).

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The major serum proteins, the immunoglobulins unfortunately are particularly hydrophobic. Storage of IgG class antibodies causes formation of aggregates, leading to even other hydrophobicity. The extent to which aggregation increases the hydrophobicity of IgG is presently under study. Hydrophobic interactions between the cross-linked proteins fixed tissue and IgG class antibodies(or their aggregates and conjugates) can be minimized by the careful characterization of reagents and by stringent observation of optimal fixing conditions. It is imperative to remember that optimal fixation may vary from tissue to tissue.

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Hydrophobic binding between tissue proteins and the antibody molecules is also influenced by the type of ions and their concentration in the diluent buffer. The lower the ionic strength of the diluent, the weaker the strength of hydrophobic interaction. The following anions and cations are arranged in order of their diminishing effect on hydrophobicity: ANIONS ; PO4 3-, SO4 2-,Cl-,NO3-, SCN- CATIONS: NH4+,K+,Na+,Ca2+

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The other possible methods to reduce hydrophobic interactions between tissue and reagent proteins include addition of detergent(eg. 0.5% Serol 0.72) or ethylene glycol to the diluent, or raising the pH of the diluent. The most widely practiced measure to reduce background due to hydrophobic interaction is the use of blocking protein either separately or added to the diluent. However, this is the only successful if the blocking protein is of the type that can compete effectively with IgG(or its aggregates or conjugates) for hydrophobic binding sites in the tissue. Separate incubation with a blocking protein is best carried out prior to application of the primary antibody. The solution should contain proteins identical to those present in the link or labeled antibody(but not those in the primary antibody), in order to prevent nonspecific binding of the secondary antibody.

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Excessive background staining due to overfixation with formalin may be avoided by post fixation with Bouins, Zenkers or B5 Fixative. Ionic (Electrostatic) Interaction Ionic interactions result when proteins of opposite netcharges meet. Because IgG class antibodies have isoelectric points(pI) ranging from 5.8 to 7.3, most will have a net negative surface charge at a buffer pH of between 7.0 to 7.8. Ionic interaction of these antibodies can be expected if tissue proteins have a net positive surface charge. Negatively charged sites on endothelia and collagen fibers were found to interact with cationic conjugates composed of rabbit Fab fragments and horseradish peroxidase type VI(pH 10.0). In general, ionic interaction can be reduced by use of diluent buffers with higher ionic strength, or by the addition of 0.1 to 0.5M NaCl to the same buffer.

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Unfortunately, most diffuse background staining is the result of a combination of ionic and hydrophobic interactions, and remedies for one type of interaction may aggravate the other type. One example of probable concurrent hydrophobic and ionic interactions is the nonspecific binding of IgG molecules to collagen and elastin. Another example is perhaps the adherence of biotinylated antibody complexes to both plastic and glass surfaces. The causes of this binding may be unfavorable conditions during biotinylation which result in antibody aggregation and reduced stability. Similar circumstances- may contribute to background staining on tissue as well.

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Endogenous Enzyme Activities For practical purposes in Immunohistochemistry, “endogenous peroxidase activity” and “pseudoperoxidase activity” can be considered the same. Peroxidase activity results in the decomposition of H2O2 and is a common property of all hemoproteins such as hemoglobin(red cells), myoglobin(muscle cells), cytochrome (granulocytes, monocytes) and catalases(liver and kidney). Hemoglobin may give rise to endogenous peroxidase acitivity which can also be encountered interstitially.

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The most frequently used procedure for the suppression of endogenous peroxidase activity in formalin-fixed tissue entails the incubation of sections in 3% H2O2 for 8-10minutes. Methonolic H2O2 treatment (1 part 3% H2O2 plus 4 parts absolute methanol) for 20min is also used, but is not recommended for specimens where cell surface markers are to be stained. Methanolic treatment may also detach frozen sections from their carrier glass. Endogenous peroxidase activity can be suppressed by a mixture of sodium azide and H2O2. It should be noted that, in most work with formalin-fixed tissue, the successful interpretation of specific staining is not impaired by the activity of endogenous peroxidase. (In cell preparations and frozen sections, routine quenching of endogenous peroxidase is advisable or an APAAP procedure should be employed, as follows).

Endogenous Peroxidase activity:

Endogenous Peroxidase activity If a specimen is rich in endogenous peroxidase activity, an enzyme label of calf intestine alkaline phosphatase instead of peroxidase is recommended. Immunoalkaline phosphatase technology does not require suppression of endogenous peroxidase activity. Quenching of endogenous tissue alkaline phosphatase activity other than that of intestine is achieved simply by including 5mM Levamisole in the substrate solution.

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Natural Antibodies Low level natural antibodies, present in the antiserum as a result of prior environmental antigenic stimulation, may increase in titer during immunization with use of adjuvants and, as a consequence, can give rise to nonspecific staining. In 1973, Osborn et al, reported that sera from nonimmunized Rabbits and goats, but not from Guinea pigs, contained environmental antibodies to keratins. This may be an example of epithelial background staining caused by natural antibodies. Although this type of staining has been observed with low dilutions of rabbit sera, attempts to isolate or remove these antibodies from the serum have not been successful. Most natural antibodies are of the nonprecipitating type(i.e. they cannot be removed by batch absorption with antigen) and occur only in relatively low concentrations. These antibodies are usually rendered non-reactive on tissue if the antiserum is used at a sufficiently high dilution or incubated for short periods.

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Contaminating antibodies Isolated antigens used for immunization are rarely pure. If the hosts immune system reacts to the impurities, contaminating antibodies will result. Usually, these contaminating antibodies are present in low concentration and will not detract from the immunohistochemical specificity of high titered antiserum as long as the antiserum is diluted properly. However, if contaminating antibodies do interfere with specificity, affinity absorption of the antiserum is usually performed. It has been found that so-called “batch-absorbed” antisera almost always contain residual levels of contaminating antibodies(mostly of the non-precipatiting type) and will cause nonspecific staining of tissue if used at high concentration. It should be kept in mind, when monitoring and evaluating the results of antiserum absorption, that such technique as immunodiffusion, immunoelectrophoresis and rocket immunoelectrophoresis can be used to exclude an antiserum as non-monospecific, but not to establish monospecificity. Monospecificity must be established by extensive work on tissues.

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Endogenous Avidin-Binding Activity(EABA) Endogenous Biotin : Biotin is a vitamin and coenzyme found in a wide variety of tissues, for example liver and kidney contain biotin bound to enzymes and other proteins. Biotin binds avidin or streptavidin specifically. It is this exceptionally strong interaction which has been utilized in Immunohistochemistry, and has led to the development of three principal methods: the labeled avidin-biotin(LAB) method, the bridged avidin-biotin(BRAB) method, and the avidin-biotin-horseradish peroxidase complex(ABC) method. Endogenous avidin-binding activity can be noted with all avidin-biotin binding techniques and is primarily due to endogenous biotin. This binding activity is most pronounced when using cryostat sections, but can be overcome by adding avidin to block endogenous biotin. Suppression of endogenous avidin binding activity, when necessary is best performed by successive 20min incubations of the tissue sections in 0.1% avidin and 0.01% biotin. Reports of endogenous avidin-binding activity include the staining of myelin in the absence of primary antibody in an ABC procedure, and the nonimmunochemical staining of mast cells in frozen and paraffin-embedded tissue. Also, Guesdon et al, found EABA in granulocytes from mouse spleen.

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Binding to Lectin-Like Substances: The avidin molecule contains 10% carbohydrates, which may bind to lectin-like substances in tissues. This binding may be blocked with an analogue to the carbohydrate on avidin. For example, 0.1M alpha-methyl-D-mannoside can be added to the solution containing avidin. Streptavidin, which does not contain sugars, does not exhibit this reaction.

Antigen Diffusion:

Antigen Diffusion When the tissue marker to be stained has diffused from its sites of synthesis or storage into the surrounding tissue, it may produce specific background staining. A typical example is the diffusion of thyroglobulin from the thyroid follicular epithelium and colloid-containing lumen into the surrounding stromal tissue. Similarly, specific background may result when the tissue marker is also present in high concentrations in blood plasma and has perfused the tissue prior to fixation. This can be seen when tonsil tissue is stained for immunoglobulins G,A,M heavy chains, and kappa and lambda light chains, particularly when fixation was not performed promptly and when the antisera used were not diluted sufficiently. Another form of specific background staining may result from the ingestion of target antigens by phagocytes, resulting in staining normally not seen in such cells.

Cross-Reactivity:

Cross-Reactivity Background staining due to cross-reactivity of the antibody monoclonal or polyclonal may result when an epitope of the tissue antigen to be stained with other proteins. A typical example is unabsorbed antiserum to carcinoembryonic antigen (CEA) which shares epitopes with several formal tissue proteins. Careful absorption of such antisera as in the case of monoclonal antibodies, careful staining of clones will eliminate the background staining. Nonspecific cross-reactivity of an antibody with similar or dissimilar epitopes on different antigens may also be the cause for confusing background. This, however, is rare and can be avoided by use of antibodies from hyperimmunized or carefully selected animals.

Receptors:

Receptors Fc Receptors are a family of detergent-soluble membrane glycoproteins with approximate molecular weights of 60-70kD. They comprise less than 1% of the total membrane proteins and are most frequently present on macrophages and granulocytes, but have also been reported on B cells and some T cells. The intrinsic affinity for the FcR portion of monomeric IgG is approximately 1*106 to 1*108M IgG, but higher for polymers and immune complexes of IgG. There are considerable class/subclass and species specificity among different Fc receptors. For exmple,Fc receptors on human cells were found to bind mouse monoclonal IgG2a and IgG3, but not other IgG subclasses. Goat sera does not react with Fc receptors of human leucocytes. Nonspecific background staining due to the lability of Fc receptor proteins is more common in frozen sections and smears than in tissues fixed by harsher procedures. It can be avoided by use of F(ab’)2 fragments instead of whole IgG molecules, and by careful screening of monoclonal antibodies. Detergent treatment of cell smears was found to eliminate Fc receptor activity on granulocytes.

Complement mediated binding:

Complement mediated binding Complement mediated binding is rarely a cause of background, because by the time large pools of antisera are added, the complement is usually inactivated.

Miscellaneous sources:

Miscellaneous sources Artifactual staining distinguished by diffuse staining of all or most tissue elements within the affected area may be caused by physical injury to tissue, by tissue drying out prior to fixation, by incomplete penetration of the fixative into the tissue, or by residual embedding medium. Necrotic areas due to autolysis of tissue may stain with all reagents.

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Buffer 0.005M Tris-buffered saline Distilled Water 10 liters Sodium chloride 80.0g TRIS(hydroxymethyl methylamine) 6.05g M HCl 44ml If necessary, adjust final pH to 7.6 with either 1M HCl or 0.2M Tris solution

Direct immunoperoxidase for either the traditional conjugated or EPOS Techniques:

Direct immunoperoxidase for either the traditional conjugated or EPOS Techniques Bring sections to TBS, drain off, incubate in non-immune serum Drain off and wipe around section 3a. Incubate in optimally diluted peroxidase-labelled primary antibody for 1-15hours.This can be achieved at either ambient temperature or 4degreeC. 3b. Incubate in the EPOS peroxidase pre-diluted primary antibody for 1-2hours at ambient temperature. Gently wash in TBS. Incubate in freshly prepared DAB solution Rinse in TBS and wash in running water Counterstain in hematoxylin, dehydrate, clear and mount

Indirect technique for monoclonal primary antibodies:

Indirect technique for monoclonal primary antibodies Bring sections to TBS. Drain off slide, wipe off excess moisture around the section Apply optimally diluted primary monoclonal antibody, 30-60min Gently wash the slides with TBS Cover with an optimally diluted peroxidase-conjugated rabbit anti-mouse containing 1/25 dilution of normal human serum. Gently wash slides with TBS Incubate in freshly prepared DAB solution for10min Rinse in TBS and transfer to running water Counterstain in hematoxylin, dehydrate, clear and mount. Modifications required for polyclonal primary antibodies Before step 3, incubate in 1/10 dilution normal swine serum for10min. This must be drained, but not washed off, before primary antibody is used. Step 5, substitute rabbit anti-mouse antibody with peroxidase-conjugated swine anti-rabbit.

Peroxidase-antiperoxidase technique:

Peroxidase-antiperoxidase technique Bring sections to TBS 2. Drain off and wipe around section 3. Incubate in optimally diluted primary antibody for 30-60min. 4. Gently wash in TBS 5. Incubate in optimally diluted rabit anti-mouse antibody for 30min. 6. Repeat step 4 7. Incubate in optimally diluted mouse peroxidase anti-peroxidase for 30min. 8. Repeat step 4 9. Incubate in freshly prepared DAB solution 10. Rinse in TBS and wash in running water 11. Counterstain in hematoxylin, dehydrate clear and mount. Modifications required for rabbit primary antibody Between steps 2 & 3 :Incubate with 1/10 dilution of normal swine serum for 10min. Drain, but do not wash off before applying primary antibody. Step 5: replace rabbit anti-mouse with swine anti-rabbit antibody Step 7: replace mouse with rabbit peroxidase anti-peroxidase.

Avidin-Biotin techniques:

Avidin-Biotin techniques Bring sections to TBS Drain and wipe off excess TBS around section Incubate in optimally diluted primary antibody for 30-40min Gently wash slides with TBS Incubate in optimally diluted biotinylated bridge reagent for 30min Repeat step 4 Incubate in optimally prepared labeled avidin or avidin-biotin complex for 30min. Repeat step 4 Incubate in DAB substrate solution Wash in running water, counterstain in hematoxylin, dehydrate clear and mount. Modifications required for rabbit primary antibody Between steps 2 & 3 :Incubate with 1/10 dilution of normal swine serum for 10min. Drain, but do not wash off before applying primary antibody Step 5: Replace with bridge reagent biotinylated swine anti-rabbit.

Alkaline-phosphatase techniques Indirect technique for polyclonal antibodies:

Alkaline-phosphatase techniques Indirect technique for polyclonal antibodies Take sections to TBS. Drain off TBS and incubate in normal swine serum, diluted 1/10with TBS, for 10min Incubate in optimally diluted rabbit primary antibody, for 30-60min Gently wash in TBS Incubate in optimally diluted alkaline-phosphatase-conjugated secondary antibody, for 30-40min Repeat step 4 Incubate in substrate solution of choice, for example fast red solution Wash in running tap water Counterstain and mount as appropriate Notes: Incorporating the following after step 5 may enhance the reaction end product. 5a. Gently wash off alkaline phosphatase-conjugate. Tip off buffer. 5b. Apply alkaline phosphatase-conjugated antibdody directed against the species of the secondary antibody.

Alkaline Phospatase anti-alkaline phosphatase(APAAP) for monoclonal antibodies :

Alkaline Phospatase anti-alkaline phosphatase(APAAP) for monoclonal antibodies Take sections to TBS. Drain off normal swine serum, and wipe off excess around the sections Incubate in primary antibody at optimal dilution, for 30-40min. Gently wash in TBS. Incubate in optimally diluted unconjugated rabbit anti-mouse bridge antibody for 30min. Incubate in alkaline phosphatase-anti alkaline phosphatase complex at the optimal dilution for 30min. Repeat step 5 Incubate in substrate medium of choice, for example fast red solution Wash in running tap water as appropriate. Notes: The reaction end-product may be enhanced by repeating steps 5-8 once or twice with a reduction of the incubation times to 10min. As the alkaline phosphatase label is usually intestinal, it is resistant to blocking with levamisole at the concentration described and hence it is included in the substrate mixture. Levamisole blocks most other types of alkaline phosphatase.

Immunogold techniques Heat mediated antigen retrieval fluids:

Immunogold techniques Heat mediated antigen retrieval fluids Citrate buffer Sodium citrate 29.4g 1M HCl 29.4g Distilled water 10 Liters Adjust pH to 6.0 using 1M HCl Tris-EDTA Tris 14.4g EDTA 1.44g 1M HCl 1ml Tween 20 0.3ml Distilled water 600ml Add this TRIS EDTA and acid to the distilled water and pH to 10 with hydrochloric acid, then add the Tween

Buffers Tris-buffereed saline containing bovine serum albumin(BSA-TBS) :

Buffers Tris-buffereed saline containing bovine serum albumin(BSA-TBS) Tris(hydroxymethyl) methylamine 12.14g Sodium chloride 45g Bovine serum albumin 5g Sodium Ozide 6.5g Distilled water 5 Liters Adjust final pH to 8.2 with 1M HCl Veronal acetate buffer Sodium acetate trihydrate 0.972g Sodium barbitone 1.472g Distilled water 247.5g 0.1M HCl 2.5ml

Indirect immunogold technique for monoclonal antibodies:

Indirect immunogold technique for monoclonal antibodies Take sections to distilled water Treat sections with Lugol’s iodine for 5min, clear with 2.5percent sodium thiosulfate and then wash well in running tap water. Take sections to BSA-TBS, drain and wipe excess around section. Incubate in normal 1/20 goat serum in BSA-TBS for 10 min. Drain, wipe off excess serum. Incubate in primary antibody optimally diluted in BSA-TBS for 30-60min. Gently wash in TBS Incubate in gold-conjugated secondary antibody at optimal dilution in BSA-TBS for 60min. Repeat step 7. Wash in 0.1M phosphatase buffered saline(PBS), pH 7.6, for three 2-min changes. Post-fix with 2% glutaraldehyde in PBS for 10-15min Wash well in several changes of distilled water and enhance staining with silver. Counterstain, dehydrate, clear and mount.

Silver enhancement:

Silver enhancement Solution:2M Citrate buffer stock. Trinatrium citrate 23.5g Citric acid 25.5g Distilled water 100ml Silver solution Silver lactate 110mg Distilled water 15ml Hydroquinone solution Hydroquinone 950mg Distilled water 15ml Gum acacia 50percent Gum acacia solution 7ml Silver enhancement solution Silver lactate solution 15ml Hydroquinone solution 15ml M Citrate buffer 10ml Distilled water 60ml 50 percent Gum acacia solution 7ml Method: Rinse the sections in 0.2M Citrate buffer for 2min Incubate the sections in freshly prepared silver enhancement solution at room temperature protected from the light. Development will take place in approximately 3min. Fix in a 1:10solution at fixing solution(Amersham international) for 2 min. Wash well in running water.

DAB METHOD Solutions:

DAB METHOD Solutions Tris-HCl buffer is recommended for DAB: 0.2M TRIS (containing 24.008g/l) 12ml 0.1N HCl 19ml Distilled water 19ml DAB solution DAB 5mg Tris-HCl buffer(pH 7.6) 10ml H2O2(freshly prepared and added just before use) 0.1ml The solutions should be used immediately after preparation. The sections are incubated at room temperature until a dark brown reaction product is obtained, usually after 5-10min. The reaction end-product resists alcohol dehydration and clearing in xylene. Modification of DAB method(employing chemical enhancement) If slides are batch-processed, the DAB may be made up in bulk and the sections stained in a staining trough. Dissolve 0.15g DAB in 10ml Tris-HCl Dissolve 0.2g imidazole in 10ml Tris-HCl Add above two solutions to 280ml Tris-HCl The sections are incubated for 30sec and then 8 drops of 30percent w/v of fresh hydrogen peroxide is added and the sections incubated for further 10min

3-Amino-9-ethylcarbazole:

3-Amino-9-ethylcarbazole 1. Dissolve 10mg of 3-Amino-9-ethylcarbazole in 6ml dimethyl sulfoxide, and then add 50ml 0.02M acetate buffer, pH 5.0-5.2 2. Add 0.4ml of 0.3% hydrogen peroxide and use immediately 3. Rinse the sections in 0.02M acetate buffer,pH 5.0, filter the substrate solution onto the sections and incubate for 5-10min, at room temperature. As the red reaction end-product is soluble in alcohol and xylene, the sections must be mounted in aqueous mounting medium.

Fast Red TR:

Fast Red TR Naphthol-AS-MX phosphate, free acid 4.0mg N,N-dimethyl formamide 0.2ml M Tris-HCl buffer, pH 8.2 9.8ml Levamisole 2.4mg Fast red TR salt 10mg Dissolve the Naphthol-AS-MX phosphate in N,N dimethylformamide and then add the Tris buffer. Add and dissolve the levamisole and fast red TR salt and immediately filter onto the sections. Incubate the sections for 10-20min, and as the bright red reaction product is soluble in alcohol, mount in an aqueous medium. A blue reaction product can be obtained by using 4mg fast blue BB instead of fast red TR salt. Counterstaining with hematoxylin would not be appropriate with blue salt.

Alternative Fast red substrate solution recommended for cytological preparations This solution described by Ponder and Wilkinson would appear to be more effective against endogenous alkaline phosphatase in pleural aspirates than the previous solution Solution:

Alternative Fast red substrate solution recommended for cytological preparations This solution described by Ponder and Wilkinson would appear to be more effective against endogenous alkaline phosphatase in pleural aspirates than the previous solution Solution Naphthol AS-BI phosphoric acid sodium salt 5.0mg N,N dimethylformamide 0.2ml Veronal acetate buffer,pH 9.2 9.8ml Levamisole 2.5mg Fast Red TR salt 5.0mg This solution prepared by dissolving the Naphthol-AS-BI in N,N dimethylformamide in a glass vial. Add the buffer and levamisole and mix. Immediately before staining dissolve the fast red salt in the substrate solution and filter before use.

Hexazotized new fuchsin:

Hexazotized new fuchsin Naphthol-AS-BI phosphate 5.0mg N,N –dimethyl formamide 60microliter M Tris-HCl buffer, pH 8.7 10ml M levamisole 10microliter 4 % sodium nitrite (freshly prepared) 50microliter 5% new fuchsin in 2M HCl 20microliter Add the new fuchsin to the sodium nitrite, mix for 30-60seconds and then add the Tris buffer and levamisole. Immediately before staining add the Naphthol AS-BI phosphate, dissolved in the N,N-dimethyl formamide,and filter directly onto the sections. Incubate for 20min. The reaction end-product is bright red, but whilst the end-product is considered to be resistant to dehydration, clearing in xylene and mounting in resinous mounting media, it is not always consistent. Therefore it is advisable to water-mount. A modified new fuchsin method proposed by Stein et al in 1985 is more complex than the first, but many users agree that the final reaction products is very more intense.

Modified new fuchsin method:

Modified new fuchsin method Solution 1 M 2-amino-2-methyl-1,3-propanediol 18ml M Tris-HCl buffer, pH 9.7 50ml Sodium chloride 600mg Levamisole 28mg Solution 2 Naphthol AS-BI phosphate 35mg N,N-dimethylformamide 0.42ml Dissolve the Naphthol AS-BI phosphate in N,N dimethylformamide. Solution 3 New Fuchsin(5g in 100ml 2N HCl) 0.14ml Sodium nitrite(freshly prepared. 40mg in 1ml distilled water) 0.35ml Mix the new fuchsin with the freshly prepared sodium nitrite and incubate in the mixture for 60 seconds at room temperature whilst agitating. Mix solutions 1 and 2 and then add solution 3. Adjust the pH to 8.7 by adding HCl. Mix well, filter through ordinary filter paper directly onto slides and incubate for 20min.

Nitro blue tetrazolium method for alkaline phosphatase:

Nitro blue tetrazolium method for alkaline phosphatase Solutions Buffer solution 0.2M Tris-HCl, pH 9.5, containing 10mM MgCl2. Solution A 5 mg 5-bromo-4-chloro-3-indolyl phosphate(BCIP) is dissolved in 0.1ml dimethyl formamide(DMF) and then in 1.0ml of the above buffer. Solution B 5 mg nitro blue tetrazolium is dissolved in 0.1ml DMF. Solutions A and B are added, with continous stirring to 30ml of above buffer and filtered. Once filtered, incubate immediately for 20min-12hours. The intense blue-black reaction product at the site of alkaline phosphatase activity is soluble in alcohol and xylene, hence aqueous mounting is recommended.

Part 1:

Part 1 1.Fixation formalin fixation and paraffin embedding 2.Sectioning 3. Whole Mount Preparation Tissue preparation

Part 2:

Part 2 1. Antigen retrieval Proteolytic enzyme method and Heat-induced method 2. Inhibition of endogenous tissue components 3% H 2 O 2 , 0.01% avidin 3. Blocking of nonspecific sites 10% normal serum pretreatment

Part 3:

Part 3 Make a selection based on the type of specimen, the primary antibody, the degree of sensitivity and the processing time required as well as the cost of the reagents. staining

Troubleshooting:

Troubleshooting 1. Weak or No Staining 2. Over-staining 3. High Background

Controls :

Positive Control It is to test for a protocol or procedure used. It will be ideal to use the tissue of known positive as a control. Negative Control It is to test for the specificity of the antibody involved. Controls

High Background :

High Background Sources Solutions Inadequate washing of sections Wash at least 3 times between steps Tissue contains endogenous enzyme Block endogenous enzyme activities Tissue contains endogenous biotin activity Block endogenous biotin activity using the avidin/biotin blocking reagent prior to incubation of primary antibodies. Non-specific binding of primary antibodies to tissue or high antibody concentration Non-specific binding may be reduced by using higher dilution of primary antibodies Non-specific binding of secondary antibodies to tissue Treat tissue with normal serum block from the same species as secondary antibodies Diffusion of tissue antigen due to inadequate fixation Increase duration of postfixation

Over-staining:

Over-staining Sources Solutions The concentration of antibodies was too high Reduce antibody concentration or perform a titration to determine the optimal dilution for primary and secondary antibodies Incubation time was too long Reduce incubation time Incubation temperature was too high Reduce incubation temperature Substrate incubation time was too long Reduce substrate incubation time Sections dried out Avoid sections being dried out

Weak or No Staining:

Weak or No Staining Sources Solutions Tissue overfixation Reduce the duration of postfixation or perform an appropriate antigen retrieval procedure Incompatible secondary and primary antibodies Use secondary antibody that will interact with primary antibody. Inactive secondary antibody or other reagents Replace with a new batch of reagents Inadequate substrate incubation time Increase the substrate incubation time Incorrect mounting medium Choose a correct mounting medium Reagents applied in wrong order or steps omitted Check notes or procedure used

Weak or No Staining :

Weak or No Staining Sources Solutions Inadequate deparaffinization Deparaffinize sections longer or change fresh xylene Inactive primary antibodies Replace with a new batch of antibodies Antibodies do not work due to improper storage Aliquot antibodies into smaller volumes and store in freezer (-20 to -70℃) and avoid repeated freeze and thaw cycles. Antibody concentration was too low Increase the concentration of antibodies. Or run a serial dilution test to determine the optimal dilution that gives the best signal to noise ratio Inadequate antibody incubation time Increase antibody incubation time Inadequate or improper tissue fixation Increase duration of postfixation or try different fixatives

Example:

Example 1. Prepare and fix tissue 2. Antigen retrieval 3.Suppress endogenous peroxidase activity 4. Block nonspecific sites in the tissues 5. Incubate the tissues with the primary antibody 6. Incubate the tissues with the secondary antibody 7. Incubate the tissues with SP 8.Add DAB and incubate until desired staining is achieved

ALGORITHMIC IMMUNOHISTOCHEMICAL DIAGNOSIS OF UNDIFFERENTIATED TUMORS & METASTATIC CARCINOMAS:

ALGORITHMIC IMMUNOHISTOCHEMICAL DIAGNOSIS OF UNDIFFERENTIATED TUMORS & METASTATIC CARCINOMAS

CURRENT DILEMMAS IN DIAGNOSTIC IMMUNOHISTOLOGY:

CURRENT DILEMMAS IN DIAGNOSTIC IMMUNOHISTOLOGY Ever-increasing number of commercial reagents for use in evaluation of human specimens; as of 12/02, there were over 2000 such antibodies listed in the Linscott catalogue Many confusing entries in the pathology literature, vis -a- vis the “specificity” (and implied usefulness) of a great many antibody reagents

“SPECIFICITY” OF IMMUNOHISTOLOGIC REAGENTS:

“SPECIFICITY” OF IMMUNOHISTOLOGIC REAGENTS Related, but not identical, to classical definition given by Galen & Gambino : Spec = True negatives/True negatives + False positives In reality, “specificity” of immunohisto -logical reagents must be evaluated in many restricted and well-defined contexts Hence, “specificity” is a relative term in this applied clinical setting

THE INTEGRATED APPROACH TO IMMUNOHISTOLOGY: USING “NON-SPECIFIC” REAGENTS TO ADVANTAGE:

THE INTEGRATED APPROACH TO IMMUNOHISTOLOGY: USING “NON-SPECIFIC” REAGENTS TO ADVANTAGE One is faced with an “either-or” decision in diagnostic immuno -histology; either use no reagents at all (since none is “absolutely” specific in the most stringent terms), or use them in particular ways that are dictated by relative antibody specificities The latter approaches do not “waste” information that is potentially gleaned from “non-specific” antibodies, and at the same time safeguard the user against erroneous interpretations

INTEGRATED ALGORITHMIC IMMUNOHISTOLOGY OF MALIGNANT TUMORS: NECESSARY CONDITIONS:

INTEGRATED ALGORITHMIC IMMUNOHISTOLOGY OF MALIGNANT TUMORS: NECESSARY CONDITIONS The user must control and standardize the processing of all tissues in his or her laboratory (e.g., fixative; fixation times; microwave-mediated antigen “retrieval”) The user must personally define optimal antibody concentrations rather than accept the recommendations of manufacturers Data must be accrued on spectra of reactivity for all antibodies used in the laboratory, either “in-house” or by adopting the EXACT methods of someone else who has already done this type of reagent analysis

INTEGRATED ALGORITHMIC IMMUNOHISTOLOGY OF MALIGNANT TUMORS: NECESSARY CONDITIONS (CONT.):

INTEGRATED ALGORITHMIC IMMUNOHISTOLOGY OF MALIGNANT TUMORS: NECESSARY CONDITIONS (CONT.) Data on antibody performance must be used as the substrate for formal statistical analysis of specificity & sensitivity in well-defined diagnostic settings, producing a “relative values” system The sequence of interpretation of panels of immunostains is governed by their relative statistical values within each predefined setting, moving from highest to lowest predictive values Discrete morphological categories must be established within which the statistical data are applied; e.g., small round-cell tumors, large polygonal-cell tumors, etc.

POTENTIALLY MORPHOLOGICALLY-INDETERMINATE TUMORS: GENERIC CATEGORIES:

POTENTIALLY MORPHOLOGICALLY-INDETERMINATE TUMORS: GENERIC CATEGORIES Small round-cell tumors Large polygonal-cell tumors Spindle-cell tumors Pleomorphic tumors Hematopoietic tumors (in subsets of the first two categories)

ALGORITHMIC IMMUNOHISTOLOGY: CHOICE OF REAGENTS:

ALGORITHMIC IMMUNOHISTOLOGY: CHOICE OF REAGENTS Panels of antibodies in each algorithm are dependent upon the generic classification of the tumor under study Several antibodies appear in more than one algorithm, but their places in the relative sequences of interpretation (or the diagnoses that positivity yields) differs from one setting to another New antibodies may be substituted for old ones or used to supplement existing reagents in all algorithms

KEY POINTS IN ALGORITHMIC IMMUNOHISTOLOGY:

KEY POINTS IN ALGORITHMIC IMMUNOHISTOLOGY Consistency of results is totally dependent upon consistency of METHODOLOGY In order to adopt the algorithms of another investigator, his or her methods MUST be followed down to the last detail or differences in antibody performance will result

POTENTIALLY UNDIFFERENTIATED SMALL CELL TUMORS:

POTENTIALLY UNDIFFERENTIATED SMALL CELL TUMORS Small cell squamous carcinoma Small cell neuroendocrine carcinoma Malignant melanoma Lymphoma Granulocytic sarcoma Neuroblastoma Primitive neuroectodermal tumor Rhabdomyosarcoma

POTENTIALLY UNDIFFERENTIATED LARGE-CELL TUMORS:

POTENTIALLY UNDIFFERENTIATED LARGE-CELL TUMORS Poorly-differentiated squamous cell carcinoma Adenocarcinoma Malignant melanoma Malignant lymphoma Granulocytic sarcoma Epithelioid-cell sarcomas Epithelioid sarcoma Clear cell sarcoma Alveolar soft part sarcoma Epithelioid malignant peripheral nerve sheath tumor Epithelioid leiomyosarcoma

POTENTIALLY UNDIFFERENTIATED SPINDLE-CELL TUMORS:

POTENTIALLY UNDIFFERENTIATED SPINDLE-CELL TUMORS Sarcomatoid squamous cell carcinoma Spindle-cell malignant melanoma Leiomyosarcoma Fibrosarcoma Malignant fibrous histiocytoma Malignant peripheral nerve sheath tumor Angiosarcoma

ANTIBODIES USED IN EVALUATION OF METASTATIC CARCINOMAS OF UNKNOWN ORIGIN:

ANTIBODIES USED IN EVALUATION OF METASTATIC CARCINOMAS OF UNKNOWN ORIGIN Keratin & Epithelial membrane antigen Prostate-specific antigen Thyroglobulin Thyroid transcription factor-1 Gross cystic disease fluid protein-15 S100 protein Carcinoembryonic antigen CA19.9 CA-125 Placental alkaline phosphatase CD15 Estrogen & Progesterone receptor proteins

ANTIBODIES APPLIED TO THE STUDY OF SMALL ROUND-CELL TUMORS:

ANTIBODIES APPLIED TO THE STUDY OF SMALL ROUND-CELL TUMORS KERATIN S100 EMA HMB45 CD45 MART-1 CD15 FLI-1 VIMENTIN SYN DES CD56 MSA CD99 MYOGENIN

ANTIBODIES APPLIED TO THE STUDY OF LARGE POLYGONAL-CELL TUMORS:

ANTIBODIES APPLIED TO THE STUDY OF LARGE POLYGONAL-CELL TUMORS KERATIN S100 VIMENTIN HMB45 BER-EP4 MART-1 CD45 CEA CD15 THROMBOMODULIN MOC31 CALRETININ CD117

REAGENTS APPLIED TO THE STUDY OF SPINDLE-CELL TUMORS:

REAGENTS APPLIED TO THE STUDY OF SPINDLE-CELL TUMORS KERATIN S100 PROTEIN EMA CD56 VIMENTIN CD31 DESMIN CD34 MSA CD57 THROMBOMODULIN CD99

REAGENTS APPLIED TO THE STUDY OF PLEOMORPHIC TUMORS:

REAGENTS APPLIED TO THE STUDY OF PLEOMORPHIC TUMORS KERATIN HMB45 EMA MART-1 VIMENTIN THROMBOMODULIN DESMIN CD31 MSA CD34 S100 PROTEIN CD99

REAGENTS USED IN PARAFFIN SECTION PHENOTYPING OF HEMATOPOIETIC TUMORS:

REAGENTS USED IN PARAFFIN SECTION PHENOTYPING OF HEMATOPOIETIC TUMORS CD15 CD79A CD20 LYSOZYME CD30 MYELOPEROXIDASE CD43 CD3 CD45 CD5 CD68 CD163

DETERMINATION OF ORIGIN FOR CARCINOMAS OF OCCULT OR CLOSELY-APPOSED SITES:

DETERMINATION OF ORIGIN FOR CARCINOMAS OF OCCULT OR CLOSELY-APPOSED SITES Use of immunohistochemistry in this setting relies on use of antigen “catalogues” for a variety of human carcinoma types Antigens are grouped into “inclusionary” & “exclusionary” categories with respect to each tumor type, and arranged algorithmically in order of relative predictive values Resulting antigen profiles constitute protein “fingerprints” of individual carcinoma types, which allow for their discrimination from one another Few tumor- specific markers are available; hence, broad antibody panels are used

EXAMPLES OF CHARACTERISTIC IMMUNOPHENOTYPES OF SELECTED CARCINOMAS:

EXAMPLES OF CHARACTERISTIC IMMUNOPHENOTYPES OF SELECTED CARCINOMAS Breast : CK+; GCDFP15+; S100+/-; CEA-; ER+/-; CA125-; CA19.9-; PLAP-; EMA+; TTF-1- Lung : CK+; GCDFP15-; S100-; CEA+; ER-; CA125-; CA19.9+/-; PLAP+/-; EMA+; TTF-1+ Kidney : CK+; GCDFP15-; S100-; CEA-; ER-; CA125-; CA19.9-; PLAP+/-; EMA+; TTF-1- Adrenal : CK-; GCDFP15-; S100-; CEA-; ER-; CA125-; CA19.9-; PLAP-; EMA-; TTF-1- Ovary : CK+; GCDFP15-; S100+/-; CEA-; ER+/-; CA125+; CA19.9+/-; PLAP+/-; EMA+; TTF-1- Germ Cell : CK+/-; GCDFP15-; S100-; CEA-; ER-; CA125-; CA19.9-; PLAP+; EMA-; TTF-1-

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CK DES/MSA CGB/SYN/CGA CD15/TAG72/MOC31 LCA EMA S100/HMB45/MART1 VIM FLI-1/SYN/CD99 DES/MSA/MYOGEN FLI-1/SYN/CGA/CD99 IMMUNOHISTOCHEMICAL DIAGNOSIS OF SMALL-CELL TUMORS + -- -- -- -- -- -- + + + -- + -- -- -- + -- + + + + PNET with divergent myogenic differentiation Neuroendocrine carcinoma (primary & secondary) Small-cell adeno- carcinoma Small-cell squamous CA Malignant lymphoma or leukemia + Malignant melanoma Technically inadequate specimen PNET (or metastatic neuroblastoma in children) Rhabdomyosarcoma Primitive neuro- ectodermal tumor (PNET) PNET

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KERATIN CD45 BER-EP4/CD15/CEA/S100 S100/TYROS/MART-1 VIMENTIN -- + -- + + -- + -- -- + IMMUNOHISTOCHEMICAL DIAGNOSIS OF MALIGNANT LARGE-CELL TUMORS Malignant melanoma Malignant lymphoma or leukemia Sarcoma Technically inferior specimen Squamous carcinoma OR Germ cell tumor Adenocarcinoma PLAP/CD117 + Seminoma --

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Keratin & Epithelial membrane antigen (EMA) Vimentin CD56/CD57 Collagen IV/CD31/CD34/Thrombomodulin Desmin/Muscle-specific actin/Alpha-Actin S100 + -- + -- + -- + -- + -- -- + IMMUNOHISTOCHEMICAL DIAGNOSIS OF SPINDLE CELL TUMORS Sarcomatoid carcinoma OR Synovial sarcoma Leiomyosarcoma/IMT/Fibromatosis/AFX Malignant schwannoma or neuroid malignant melanoma Malignant schwannoma Vascular sarcoma (or DFSP [CD34+ only ]) Technically inadequate specimen Fibrosarcoma/AFX/MFH/IMT

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Keratin & Epithelial membrane antigen Desmin /Muscle-specific actin S100 protein Collagen IV/CD31/CD34/Thrombomodulin TYROS/MART1 Vimentin + -- -- + -- + -- -- + -- IMMUNOHISTOCHEMICAL DIAGNOSIS OF MALIGNANT PLEOMORPHIC TUMORS + Malignant melanoma Sarcomatoid squamous cell carcinoma Angiosarcoma Technically inadequate specimen Atypical fibroxanthoma or MFH Malignant schwannoma or Neuroid malignant melanoma + Leiomyosarcoma

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PARAFFIN SECTION IMMUNOPHENOTYPING OF HEMATOPOIETIC MALIGNANCIES CD45 CK/S100/HMB45/MART1/PLAP CD15/CD30/CD79a/4KB5 VIM CD45RO LYSO/MPX CD20/CD79a/4KB5 CD43 CD163 -- + -- + + -- -- + + -- -- + -- -- + -- + Non-hematopoietic Neoplasm or Langerhans’ Cell Histiocytosis (Positivity for CD1a) Hodgkin’s Disease vs. Large-cell Non-Hodgkin’s ML Technically-inadequate Specimen Probable Sarcoma T-cell Lymphoma Granulocytic Sarcoma Non-Hodgkin’s ML, Not Otherwise Specified “True Histiocytic” Neoplasm + B-cell Lymphoma

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Seminoma (PLAP+; CD117+; VIM + ) or Adrenocortical CA (VIM+; Inhibin+; PLAP-) KERATIN Mixture PSA/TGB/TTF1 PSA+/TGB-/TTF1- Prostate CA PSA-/TGB+/TTF1- Thyroid CA PSA-/TGB+/TTF1+ Thyroid CA PSA-/TGB-/TTF1+ Lung CA All 3 (-) GCDFP CEA-M S100 PLAP CA-125 CA19-9 CK20 ER GCDFP+ alone-- Breast, salivary gland CEA+ alone-- GI tract, lung, Mullerian tract, bladder S100+ alone-- Mullerian tract, breast, salivary gland PLAP+ alone-- Mullerian tract, GI tract, lung, kidney, breast CA125+ alone-- Mullerian tract, biliary tract, pancreas, lung, breast, GI tract, liver CA19-9+ alone-- GI tract, pancreas, Mullerian tract, bladder, lung CK20+ alone-- GI tract, Mullerian tract, pancreas, lung ER+ alone-- Breast, Mullerian tract, bladder liver, stomach GCDFP+CEA+-- Breast, salivary gland GCDFP+S100+-- Breast, salivary gland GCDFP+PLAP+-- Breast GCDFP+CA125+-- Breast, salivary gland GCDFP+CA19-9+-- Salivary gland, breast GCDFP+CK20+-- Not described in literature GCDFP+ER+-- Breast CEA+S100+-- Breast, salivary gland, Mullerian tract, stomach, biliary tract CEA+PLAP+-- GI tract, biliary tract, lung, Mullerian tract CEA+CA125+-- Mullerian tract, biliary tract, pancreas, lung, GI tract, breast CEA+CA19-9+-- GI tract, pancreas, bladder, Mullerian tract, lung CEA+CK20+-- GI or biliary tracts, pancreas, endocervix, bladder, lung CEA+ER+-- Breast, stomach, Mullerian tract S100+PLAP+-- Mullerian tract, breast, stomach, kidney S100+CA125+-- Mullerian tract, breast, biliary tract S100+CA19-9+-- Mullerian tract, biliary tree, stomach, kidney S100+CK20+-- Mullerian tract, biliary tree, stomach S100+ER+-- Breast, Mullerian tract PLAP+CA125+-- Mullerian, biliary, or GI tracts PLAP+CA19-9+-- Biliary, Mullerian, or GI tracts; lung PLAP+CK20+-- Biliary,GI, or Mullerian tracts; pancreas PLAP+ER+-- Breast, Mullerian tract ALL NEGATIVE-- Renal cell carcinoma (RCC) or Germ cell tumor (rare examples) or Nasopharyngeal carcinoma (RCC is positive for CD10 and/or RCC-Antigen) IMMUNOHISTOLOGICAL EVALUATION OF MALIGNANT EPITHELIAL TUMORS OF UNKNOWN ORIGIN + -- + -- B72.3/MOC-31 CALRET/CD141 + Mesothelioma VIM -- -- + NPC HCC + -- Adrenocortical CA (Also Inhibin+) Embryonal CA (Also CD30+) EMA + PLAP RCC or NPC + -- PLAP + Embryonal CA (Also CD30+) -- EMA Either or both Both -- Either or both

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NEOPLASIA: new abnormal growth A neoplasm “ is a abnormal purposeless mass of tissue, the growth of which exceeds and is uncoordinated with that of normal tissues, and which persists in the same excessive manner after cessation fo the stimuli which evoked the change” Tumor= swelling. Benign tumor -- no infiltration into surrounding tissue. Malignant tumor = cancer Cancer is the common term for all malignant tumors. Cancer derives from the Latin term crab presumably because it “ adheres to any part that it seizes in an obstinate manner like the crab”

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Benign tumors: fibroadenomas, polyps of the colon, lipomasCARCINOMAS: -Malignant tumors of epithelial cells -well differentiated, moderately differentiated, poorly differentiated -squamous carcinomas - adeno-carcinomas alveolar papillary tubular (anaplastic, undifferentiated, large cell, small cell) (hepatocellular carcinoma, cholangiocarcinoma)

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SARCOMAS: Malignant tumors of supporting tissue -chondrosarcomas--cartilage -osteosarcomas--bone -hemagiosarcomas--blood vessel -gliomas (astrocytoma, glioblastoma) -lymphomas -melanomas -rhabdomyosarcomas -leiomyosarcomas -fibrosarcomas -seminoma, teratoma, etc.

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IMMUNOHISTOCHEMISTRY is an important adjunct to histopathologic evaluation Epithelium: Keratins --pan-keratin and antibodies to keratins of different molecular weights Supporting connective tissues: --Vimentin--fibroblasts, blood vessels --vWF, CD31 (PECAM)-- endothelial cells of blood vessels Hematopoeitic tissues: CD45, B220, CD3, F480, Mac-1, Gr-1, CD41 Muscle: desmin, smooth muscle actin Neural: GFAP, NeuN, F480/Mac-1, MBP, NSE, S100 Hormones: specific antibodies--insulin, casein, etc. Germ cells: alpha-feto protein (teratomas) Proliferation markers-Ki-67

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Caption: Picture 1. Oral malignant melanoma. Man with an ulcerated, blue-black, slightly elevated lesion in the edentulous, posterior maxilla on the right side. The lesion extends across the residual alveolar ridge onto the palate and onto the facial aspect of the ridge. The diagnosis is oral melanoma.

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Oral malignant melanoma. Japanese male patient with extensive, black-pigmented and irregularly bordered macule in the maxillary labial mucosa and midline facial gingiva, (teeth 8 and 9). (The patient's fingers are depicted.) The diagnosis is oral melanoma. Courtesy Dr Bob Goode, Tufts University

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Leukoplakia Lesions

PALATAL LESIONS OF LEUKOPLAKIA:

PALATAL LESIONS OF LEUKOPLAKIA

LEUKOPLAKIA:

LEUKOPLAKIA

References:

References Bancroft histological procedures Ihc world.co.in Culling histological procedures

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Thank you !

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