PowerPoint Presentation: MUCIN STAINING INTRODUCTION: INTRODUCTION Mucins are high molecular weight glycoproteins that are found dispersed throughout the epithelia of the-gastrointestinal, respiratory and reproductive tract. Mucins are composed of a central protein core with multiple chains of carbohydrates (polysaccharides) attached. Types of mucopolysaccharides: Types of mucopolysaccharides Neutral These can be found in glands of the GI tract and in prostate Acid (simple, or non- sulfated ) Are the typical mucins of epithelial cells containing sialic acid. Acid (simple, mesenchymal ) These contain hyaluronic acid and are found in tissue stroma . Acid (complex, connective tissue) Found in tissue stroma , cartilage, and bone and include substances such as chondroitin sulfate or keratan sulfate . DEMONSTRATION OF MUCIN: DEMONSTRATION OF MUCIN Diagnosis of poorly differentiatd adenocarcinoma Identify premalignant lesion on stomach Diagnosis of mucopolysaccharidoses STAINING TECHNIQUES: STAINING TECHNIQUES Mucicarmine oldest techniques for the detection of mucins . evaluation of acid mucins particularly those of the gastrointestinal tract and for staining the capsule of the fungus Cryptococcus neoformans . The active dye molecule found in the mucicarmine stain is a chelate complex formed between cationic aluminum ions and carminic acid. Colloidal iron ("AMP") Iron particles are stabilized in ammonia and glycerin and are attracted to acid mucopolysaccharides . Requires formalin fixation. Phospholipids and free nucleic acids may also stain. Tissue can be pre-digested with hyaluronidase to provide more specificity STAINING TECHNIQUES: STAINING TECHNIQUES Alcian blue The pH of this stain can be adjusted to give more specificity. large planar pthalocyanine molecule with a copper atom in the center. The molecule also contains four basic isothiouronium groups which carry a positive charge PAS ( Peroidic acid-Schiff) the most versatile and widely used of the techniques for the demonstration of glycoproteins , carbohydrates and mucins . Stains glycogen as well as mucins , but tissue can be pre-digested with diastase to remove glycogen. The PAS technique is based upon the reactivity of free aldehyde groups within the monosaccharide units with the Schiff reagent to form a bright red magenta end product. MUCICAMINE STAIN : MUCICAMINE STAIN PURPOSE: To stain acid mucopolysaccharides ( mucin ), which is a secretion produced by a variety of epithelial cells and connective tissue cells. Excess mucin is secreted by epithelial cells in certain inflammations and in certain intestinal carcinomas. The mucicarmine technique is also useful in determining the site of a primary tumor in that finding mucin positive tumor cells MUCICAMINE STAIN: MUCICAMINE STAIN PRINCIPLE: Aluminium is believed to form a chelation complex with the carmine changing the molecule to a positive charge allowing it to bind with the acid substrates of low density such as mucins . MUCICAMINE STAIN: MUCICAMINE STAIN SOLUTIONS: MUCICARMINE SOLUTION: Carmine, alum lake 1.0gm Aluminium hydroxide 1.0gm 50% Alcohol 100ml Aluminium chloride, anhydrous 0.5gm METANIL YELLOW SOLUTION: Metanil yellow 0.25gm Distilled water 100ml Glacial acetic acid 0.25ml MUCICAMINE STAIN: MUCICAMINE STAIN PROCEDURE: Sections is deparaffinize and hydrate to distilled water. Mayer’s hematoylin for 10 minutes. Wash in running tap water, 5 minutes. Mucicarmine solution, microwave HI power for 45 seconds. Rinse quickly in distilled water. Metanil yellow 30 seconds to 1 minutes. Dehydrate quickly in three changes of absolute alcohol. Mount and put coverslip . MUCICAMINE STAIN: MUCICAMINE STAIN RESULTS: COLONIC MUCOSA Mucin - deep rose Nuclei- black Other tissue elements- yellow MUCICAMINE STAIN: MUCICAMINE STAIN The cytoplasm of the cells lining this neoplastic gland in a colonic adenocarcinoma are pink with the mucicarmine stain, indicative of mucin production typical for an adenocarcinoma at this site. MUCICAMINE STAIN: MUCICAMINE STAIN The goblet cells of the gastrointestinal tract are filled with abundant acid mucin and stain pale blue with this Alcian blue stain. MUCICAMINE STAIN : MUCICAMINE STAIN FUTURE OF MUCIN STAINING diagnosis of adenocarcinoma , particularly poorly differentiated adenocarcinoma generally assumed that cancer ' mucin ' is the counterpart (albeit differing qualitatively) of the normal secretions of mucinous cells of crypts, ducts or acini mucin secreting adenocarcinomas may arise in tissues or organs that do not normally secrete mucin , such as prostate, breast and ovary COLLOIDAL IRON (AMP): COLLOIDAL IRON (AMP) PURPOSE : colloidal iron technique may be used in combination with the periodic acid – Schiff technique. PRINCIPLE : The bound ferric ions are detected or visualized by subsequent treatment with potassium ferrocyanide to form bright blue deposits of ferric ferrocyanide or Prussian blue COLLOIDAL IRON (AMP): COLLOIDAL IRON (AMP) SOLUTIONS : Neutral red Acetic acid, 2M (12%) Potassium ferrocyanide 2% aqueous Hydrochloric acid 2% aqueous Rhinehart & Abu'l Haj's colloidal iron suspension Working colloidal iron Colloidal iron suspension 1 vol Acetic acid, 2M 1 vol Perls ' solution 2% potassium ferrocyanide 1 vol 2% hydrochloric acid 1 vol COLLOIDAL IRON (AMP): COLLOIDAL IRON (AMP) PROCEDURE : Bring sections to water via xylene and ethanol. Place into the working colloidal iron for 15-20 minutes. Wash with distilled water. Wash with running tap water for 5 minutes to remove all traces of colloidal iron Wash with distilled water. Place into freshly made Perls ' solution for 10 minutes. Wash with distilled water. Counterstain with neutral red for 1 minute. Dehydrate with ethanols . Clear with xylene and mount with a resinous medium. COLLOIDAL IRON (AMP): COLLOIDAL IRON (AMP) RESULTS : Acid mucopolysaccharides – blue Nuclei – red Dermal mucinosis PROBLEMS AND WAYS OVERCOMING IT: PROBLEMS AND WAYS OVERCOMING IT Since this method depends on the staining of iron compounds with the prussian blue reaction, any hemosiderin present will also be stained. If this is a concern, a control section should be stained which has not been treated with colloidal iron. Material stained blue in both sections should be discounted. Nuclear fast red may also be used as a nuclear counterstain , or a Feulgen's nucleal reaction may be applied before step 2, in which case the nuclear counterstain should be omitted. A PAS may be applied following step 7, in which case the colour of the nuclear counterstain should be changed, perhaps with a strictly progressive hemalum . Acid mucosubstances will be stained blue in contrast to red neutral mucosubstances . However, they are often present as mixtures and the contrast may not be clear. ALCIAN BLUE: ALCIAN BLUE PURPOSE : Alcian blue stains acid mucosubstances and acetic mucins . Excessive amounts of non-sulfated acidic mucosubstances are seen inmesotheliomas , certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis ALCIAN BLUE: ALCIAN BLUE PRINCIPLE : Alcian blue is a group of polyvalent basic dyes that are water soluble. The blue color is due to the presence of copper in the molecule. The 3% acetic acid solution (pH2.5), Alcian blue stains both sulfated and carboxylated acid mucopolysaccharides and sulfated and carboxylated sialomucins ( glycoproteins ). It is believed to form salt linkages with the acid groups of acid mucopolysaccharides . ALCIAN BLUE: ALCIAN BLUE SOLUTIONS : 3% Glacial Acetic Acid Acetic acid 3.0 ml Distilled water 100.0 ml Alcian Blue Solution 3% glacial acetic acid 100.0 ml Alcian blue 8GX 1.0 gm Nuclear Fast Red ( Kernechtrot ) Aluminum sulfate 25.0 gm Distilled water 500.0 ml Nuclear fast red 0.5 gm ALCIAN BLUE: ALCIAN BLUE PROCEDURE : 1. Hydrate slides to distilled water. 2. 3% acetic acid, 3 minutes. 3. * Alcian blue solution, microwave: Hi power, 30 seconds. 4. Wash in running water for 2 minutes, rinse in distilled. 5. Nuclear-fast red, 5 minutes, wash in tap water. 6. Dehydrate, clear, and coverslip . *Conventional Method: Alcian blue, room temperature for 30 minutes ALCIAN BLUE: ALCIAN BLUE Nuclei (using nuclear fast red) – reddish pink Acid mucins / mucosubstances – Blue RESULTS : PAS (Periodic Acid-Schiff): PAS (Periodic Acid-Schiff) PURPOSE : The PAS technique is perhaps the most versatile and widely used of the techniques for the demonstration of glycoproteins , carbohydrates and mucins . PRINCIPLE : This is a combined method utilising the properties of both the PAS (see PAS) and Alcian blue (see Alcian blue) methods to demonstrate the full complement of tissue proteoglycans . By first staining all the acidic mucins with Alcian blue, those remaining acidic mucins which are also PAS positive will be chemically blocked and will not react further during the technique. Those neutral mucins which are solely PAS positive will subsequently be demonstrated in a contrasting manner. Where mixtures occur, the resultant colour will depend upon the dominant moiety. PAS (Periodic Acid Schiff): PAS (Periodic Acid Schiff) SOLUTIONS : 1. 1% Alcian blue in 3% acetic acid (see Alcian blue) 2. 0.5% aq periodic acid 3. Schiff's reagent (see PAS) PAS (Periodic Acid – Schiff): PAS (Periodic Acid – Schiff) PROCEDURE : Bring sections to distilled water. Stain with Alcian blue 15 mins Wash well in running tap water 2 mins Rinse in distilled water Treat with periodic acid 5 mins Wash well in distilled water Stain with Schiff's reagent 10 mins Wash well in running tap water 5 mins Stain nuclei with haematoxylin 1 min Wash in running tap water 2 mins Differentiate with acid alcohol Wash and blue nuclei in Scott’s tap water Wash in water Dehydrate, clear and mount PAS (Periodic Acid Schiff): PAS (Periodic Acid Schiff) RESULTS : acidic mucins ............................... blue neutral mucins ............................. magenta mixtures of above ......................... blue/purple nuclei ....................................... deep blue Inclusion body of tissue COMPARISON WITH H&E STAIN: COMPARISON WITH H&E STAIN Normal tissue H&E stain (Tonsil) Abnormal tissue mucin stain (Tonsil) PowerPoint Presentation: THANK YOU….