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Methods to study Histology :

Methods to study Histology Kimboka A


Cell Tissue Organ Organ system Homeostasis Histology

How to get the histology slides?:

How to get tissues for study Steps in tissue preparation Fresh tissues from the body 1. fixation Formalin ( 10% formaldehyde) Osmium tetroxide for EM Mechanism - Forms cross links with proteins (Lysine) 2. Embedding – gives support for tissue slicing Paraffin or plastic resin 3. Washing & dehydration (dehydration by graded alcohols in ascending order) 4. clearing – to remove paraffin & alcohol By xylol or tulol 5. block making How to get the histology slides?

How to get the histology slides?:

6. section cutting – 5-10 μ thick sections with microtome 7. mounting – on glass slide ( adhesive – albumin) 8. clearing – xylol / tulol 9. rehydrate – alcohols in descending order Staining nuclear stain – Hematoxylin ( basic stain & water soluble) counter stain – Eosin ( less water soluble but soluble in alcohol) – dehydrate in ascending order 10. Clearing – xylol / tulol 11.Mounting medium – cover glass How to get the histology slides?

Special situations:

Staining – routine stain – H&E Some structures are seen/ preserved (large molecules like nucleoproteins, cytoskeleton proteins, ECM proteins- collagen, membrane proteins) some are not seen/lost (small molecules -t-RNA, large molecules like glycogen & Proteioglycans are dissolved, )during the fixation/staining process Special fixatives to retain membrane ( phospholipids) Permanganate & osmium – for EM For Elastic fibers – Orcein/ Resorcin – Fuscin For reticular fibers – Silver impregnation Histochemistry & Cytochemistry Specific binding of dye with particular molecule Fluorescent dye labeled antibody to cell component Enzyme activity Autoradiography – radio isotopes tagged with precursors of a molecule  molecule incorporated into cell/ tissue before fixation Special situations

Basis of staining:

Basis of staining ACIDIC DYES BASIC DYES Eosin Hematoxylin /Methylene blue Carry net negative charge Carry net positive charge React/bind with cationic components of the cell/tissue With anionic components of cell/tissue Less specific (as compared with basic dyes) Highly pH specific Acidophilic / Eosinophilic (cytoplasmic filaments, intracellular membranous components, extracellular fibers) Basophilic substances ( Po4 of Nucleic acids, So4 of MPS, CO proteins)

What is special about Hematoxylin? :

Mostly resembles basic dye but it is a mordant (helps to form links between tissue fragment & the dye) It will not dissociate in sequential staining process  unlike other basic dyes What is special about Hematoxylin?


What is it ?  Absorb certain wavelength of light and emit different wavelength Why Metachomasia ?  Polyanions of tissues bind with dye molecules result in polymer or dimers of dye molecules  appear as different color rather than expected ( methylene blue gives red or purple color) What are metachromatic substances?  Ionized So4, Po4 of cartilage Where you find it?  Mast cell granules (heparin) & rER of Plasma cells Metachomasia

PAS =Periodic Acid Schiff:

Special stain PAS positive substances  Carbohydrate (glycogen) or carbohydrate rich molecules, Basement membrane, reticular fibers Periodic acid cleaves bond between carbon atoms  form aldehyde group Aldehyde binds with Schiff to produce magenta or pink color PAS = P eriodic A cid S chiff

Feulgen stain for Nuclear Proteins:

Acid hydrolyses or cleaves proteins from deoxyribose of DNA  leads to opening of sugar group & formation of aldehyde Schiff binds and gives magenta color to aldehyde Can be useful to quantify amount of DNA ( by using spectrophotmetry of Feulgen stained tissue) Feulgen stain for Nuclear Proteins Why RNA cannot be stained by Feulgen?

Enzymatic digestion :

For the confirmation of specific substances Pretreatment of sections with specific enzymes Diastase/amylase  for glycogen DNA ase  for DNA Enzymatic digestion

Enzyme Histochemistry:

Localization of enzymatic activity in tissues Best fixation – mild aldehyde ( formalin) Basis – localized reaction production of enzyme activity Used for acid & alkaline phosphatase, ATP ases AB (substrate) + T (trap) AT ( reaction product) + B (Hydrolyzed component of substrate) Enzyme Histochemistry enzyme

Immuno Histo Chemistry (IHC):

Antibody ( Immunoglobulin) conjugated with fluorescent dye( most common is Fluorescein) + Antigen ( foreign protein) Fluorescein  absorbs UV light and emits green fluorescence  can be seen under Fluorescent microscope ( IF - I mmuno F luorescence) Example : - actin (Antigen) of Rat  infected to Rabbit  blood of Rabbit ( have poly - clonal antibodies for Rat’s actin/ anti rat actin antibodies)  bind with Fluorescent dye I mmuno H isto C hemistry ( IHC )

Monoclonal Antibodies:

Specific antigen (actin of rat) Monoclonal Antibodies Multiple Myeloma pts . Monoclonal B ells Hybridoma cells ↓ Single specific type of antibodies ( Monoclonal ) ( against Actin) B lymphocytes of Immunized rabbit ↓

Clinical Significance of Monoclonal Antibodies :

Diagnosis of tumors(tumor markers) & Infections( HIV, Infectious Mononucleosis) Classify sub – types (B -cell and T- cell lymphomas) Treatment – Anti-TNF- α antibodies in inflammatory disorders Clinical Significance of Monoclonal Antibodies

Immunological Methods:

Immuno -fluorescence Direct (one step, less sensitive) & Indirect ( more sensitive, Expensive, labor intensive, can’t easily run in automated) methods Immunoperoxidase method Enzyme is used ( horse raddish peroxidase) to color colorless substrate into colored insoluble product Immunological Methods

Other Methods:

Other Methods Hybridization: for localizing mRNA/DNA (NA) In Situ Hybridization: Binding ( Probe + NA) in cell/tissue FISH: If Fluorochrome is used in Hybridization technique Autoradiography: by tagging the precursor molecules (Amino acids) followed by synthesis of large molecules (NA)  localize the particular tagged molecule

Microscopy :

Microscopy Resolution/ Resolving power ( RP ): the distance by which two objects must be separated to be seen as two objects RP of Unaided Human retina : 0.2 mm Light Microscope (LM) : 0.2 μ Electro Microscope (EM) : 1.0 nm LM : we see only two dimensional pictures, orientation of cut gives different patterns Artifacts: error in preparation process

orientation of cut:

orientation of cut

Three dimensional picture:

Three dimensional picture How you get it?

Types & Advantages of Microscopes:

Types & Advantages of Microscopes 1. Phase contrast M: can see live (unstained) tissue Light passing thru denser tissue of higher refractory index  out of phase from the rest  look darker Uses : identify cells in tissue cultures Modification: Interference M: quantification of tissue masses helps in study of surface properties of cells What happens to the tissues during routine staining process?

Types & Advantages of Microscopes:

Types & Advantages of Microscopes 2. dark Field M: special condenser illuminates specimen with strong oblique light Uses: In auto radiography Study crystals in urine Study microbes- slender spirochetes ( * Treponema pallidum ) 3. Fluorescent M: emits light in visible range when exposed to UV light Technique: filters are used between light source & specimen Naturally fluorescent substances: Vitamin “A”, Neuro- transmitters Uses Tracing pathways of nerve fibers, To detect growth markers of mineralized tissues *What is the disease caused by this bug?

Types & Advantages of Microscopes:

Types & Advantages of Microscopes 4. Confocal scanning M: Con jugate with focal point of lens Computer software reconstitutes the image from the data Major difference from LM: addition of detector aperture (pin hole) Uses: can see 3D pictures 5. ultra violet M: Depends on absorption of UVL by specimen Results are recorded photographically (can’t be seen directly – why?) Uses Study of nitrogen bases ( in NA) Study amount of DNA/RNA in cells  * Clinically helps in study of ploidy in tumors Highly aneuploid tumor  What is its Significance ?

Types & Advantages of Microscopes:

Types & Advantages of Microscopes 6. Polarizing M: only difference is polarizer (polarizing filter) Birefringence: ability of crystalline or Para - crystalline material to rotate the phase of polarized light (double refraction) Skeletal muscle & Leydig cells * Amyloid protein: apple green ± Uric acid: negative ± ± Ca++ pyrophosphate * ,±, ± ±  clinical Significance ?

Types & Advantages of Microscopes:

Types & Advantages of Microscopes 7. Electron M ( EM ): specimen is in vacuum Types: Transmission ( TEM ), scanning ( SEM ) Mechanism: similar to LM except that beam of electrons replace light source Recording: photoelectric plate or video detector Specimen preparation: Fixation: Glutaraldehyde ( cross links with proteins), Osmium tetroxide (reacts with * phospholipids ) makes cell/tissue electron dense for image enhancement Other steps are same as routine tissue processing except Plastic is used for embedding ± Diamond knives are used in microtome ( not metal knives) To study membranes – Freeze fracture technique {-160 °C with glycerol (to prevent ice crystal formation)} * Where you find ? ± why diamond knives are used in EM?

Types & Advantages of Microscopes:

Types & Advantages of Microscopes 7. Scanning ( SEM ) It differs from TEM that electron beam passes across the surface of spectrum (not thru specimen as in TEM) Resembles Television Can see 3D pictures 8. Atomic Force M: most powerful tool to study surface topography Non – optical M: works like finger tip Has highest resolution power – 50 pm * Specimen need not be in vacuum *what is the additional advantage?

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