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Carbs & Amyloid
Staining of carbohydrates and Amyloid in the histology lab
| Question | Answer |
|---|---|
| Neutral polysaccharides (non ionic homoglycans) | 1. Glucose containing: glycogen, starch, cellulose 2. N-acetyl-glucosamine- containing chitin |
| Neutral polysaccharides | PAS stain positive |
| Neutral polysaccharides | Negative staining for Alcian blue, colloidal iron, mucicarmine |
| Acid mucopolysaccharides (Anionic heteroglycans) | 1. Carboxylates (COOH): hyaluronic acid, found in connective tissue & umbilical cord 2. Sulfated (OSO3H) & Carboxylated (COOH)- cartilage, cornea, blood vessels, skin, lung, aorta 3. Sulfated only (COOH free)-human aorta and bovine cornea |
| PAS negative | Acid mucopolysaccharides |
| Alcian blue, colloidal iron, and mucicarmine positive | Acid mucopolysaccharides |
| Glycoproteins (mucins, mucoid, mucoprotein, mucosubstances) | 1. Neutral: egg white, stomach mucin, Paneth granules 2. Carboxylated (COOH): submaxillary glands, small intestinal mucins, fetal, sublingual gland 3. Sulfated (OSO3H) and Carboxylated (COOH) COlonic mucins |
| Can be PAS positive but not always | Glycoproteins |
| Glycolipids | 1. Cerebrosides 2. Phosphatides: PAS positive non carbohydrate containing lipid |
| PAS | Demonstration of polysaccharides, neutral mucosubstances, and basement membranes |
| PAS | 10% NBF or Bouin |
| PAS | 4-5 micrometers |
| PAS control | Kidney, liver, or cervix |
| How do you test the quality of Schiff reagent | Place 10mL of 37-40% formaldehyde in a beaker and add a few drops of Schiff reagent Reddish purple: Good Deep blue purple: Bad |
| PAS results | Rose: Glycogen, neutral mucosubstances, epithelial sulfomucins and sialomucins, colloid material of the thyroid and pars intermedia of the pituitary, basement membranes, and fungal walls |
| Not recommended fixative for PAS | Glutaraldehyde (Can produce false results) |
| PASD | Demonstration of glycogen in tissue |
| PASD | Diastase and alpha amylase act on glycogen to depolymerize it into smaller sugar units that are washed out of the section. |
| PASD | 10% NBF, formali alcohol, or absolute alcohol |
| PASD | 4-5 micrometers |
| PASD control | Liver or cervix |
| PASD results | Rose: glycogen without digestion Colorless: with digestion |
| Carmine | Demonsttration of glycogen |
| Carmine | Absolute alcohol preferred; Carnoy and Bouin ok |
| Carmine | 4-5 micrometers |
| Carmine control | Liver |
| Carmine | 1. Harris Heme 2. Wash 3. Camine solution 4. Differentiating solution 5. Rinse 80% alcohol 6. Dehydrate 7. Clear |
| Carmine results | Glycogen: pink to red Nuclei: Blue |
| Mayer mucicarmine | Staining of epithelial mucin in tissue |
| Mayer mucicarmine | Stains carboxylated and sulfated mucins |
| Mayer mucicarmine | Commonly used to identify adenocarcinomas |
| Mayer mucicarmine | 10% NBF |
| Mayer mucicarmine | 4-5 micrometers |
| Mayer mucicarmine control | unautolyzed colon, small intestine, or appedix |
| Mayer mucicarmine | 1. Weigert heme 2. Wash in water 3. stain with mucicarmine solution 4. rinse with water 5. stain with metanil yellow 6. dehydrate 7. Clear |
| Mayer mucicarmine results | Mucin: rose to red Capsule of Crytoccocus: rose to red Nuclei: black Other tissue elements: blue or yellow |
| Mayer mucicarmine | Useful for the demonstration of Cryptococcus neoformans |
| Alcian blue pH 2.5 | Demonstration of acid mucopolysaccharides |
| Alcian blue pH 2.5 | Believed to form salt linkages with the acid groups of acid mucopolysaccharides |
| Alcian blue pH 2.5 | 10% NBF or Bouin |
| Alcian blue pH 2.5 | 4-5 micrometers |
| Alcian blue pH 2.5 | 1. 3% acetic acid 2. Alcian blue 3. rinse in 3% acetic acid 4. wash in tap water 5. rinse in distilled water 6. counterstain 7. dehydrate 8. Clear |
| Alcian blue pH 2.5 results | Weakly acidic sulfated mucosubstances, hyaluronic acid, and sialomucins: Dark blue Background: pink to red Nuclei: Red |
| Alcian blue pH 1.0 | Demonstration of sulfated mucosubstances |
| Alcian blue pH 1.0 | 10% NBF or Bouin solution |
| Alcian blue pH 1.0 | 4-5 micrometers |
| Alcian blue pH 1.0 control | Unautolyzed small intestine, appendix, or colon |
| Alcian blue pH 1.0 results | Sulfated mucosubstances: pale blue Background: pink to red Nuclei: Red |
| Alcian blue with hyaluronidase | To differentiate epithelial from connective tissue mucins |
| Alcian blue with hyaluronidase | Staining will disappear or be reduced when tissue sections containing hyaluronic acid, chondroitin sulfate A, or chondroitin sulfate C are digested with testicular hyaluronidase. Glycoproteins are not affected |
| Alcian blue with hyaluronidase | 10 NBF |
| Alcian blue with hyaluronidase | 4-5 micrometers |
| Alcian blue with hyaluronidase control | umbilical cord, small bowel, appendix, or colon |
| Alcian blue with hyaluronidase | 1. Place in digestion 2. rinse 3. 3% acetic acid 4. Alcian blue 5. wash 6. rinse in distilled water 7. counter stain 8. wash 9. dehydrate 10. clear |
| Alcian blue with hyaluronidase results | Without digestion, acid mucopolysaccharides and sialomucins: Deep blue With digestion, mucosubstances containing hyaluronic acid and chondroitin: Marked loss of staining |
| Alcian blue-PAS heme | Differentiate between neutral and acidic mucosubstances |
| Alcian blue-PAS heme | 10% NBF or Zenker |
| Alcian blue-PAS heme | 4-5 micrometers; kidney should be 2-3 micrometers |
| Alcian blue-PAS heme control | kidney, or cervix |
| Alcian blue-PAS heme results | Exclusively acid mucosubstances: blue neutral polysaccharides: magenta Certain substances will be colored by both PAS and Alcian blue: Purple |
| Muller-Mowry colloidal iron | To demonstrate carboxylated and sulfated mucopolysaccharides and glycoproteins |
| Muller-Mowry colloidal iron | Colloidal ferric ions are, at a low pH, absorbed principally by carboxylated and sulfated mucosubstances. The excess reagent is washed out and the classic Prussian blue reaction is used to demonstrate iron bound to tissue |
| Muller-Mowry colloidal iron | 10% NBF, Carnoy, or alcoholic formalin Avoid chromate fixativve |
| Muller-Mowry colloidal iron | 4-5 micrometers |
| Muller-Mowry colloidal iron control | Unautolyzed small bowel, appendix, colon |
| Muller-Mowry colloidal iron | 1. 12% acetic acid 2. Ferrocyanide hydrochloric acid solution 3. wash 4. counter stain 5. wash 6. dehydrate 7. clear |
| Muller-Mowry colloidal iron results | Acid mucopolysaccharides and sialomucins: deep blue Nuclei: pink-red Cytoplasm: Pink |
| Alkaline Congo Red | Demonstration of amyloid tissue |
| Congo red | Green bifringence |
| Congo red | Alcohol or Carnoy solution preferred, 10% NBF, Bouin, or Zenker |
| What decreases staining intensity in Congo red? | Prolonged storage in 10% NBF |
| Congo red technique | 8-10 micrometers |
| Congo red control fixative | A tissue containing amyloid |
| Congo red procedure | 1. Harris Heme 2. wash 3. Alkaline salt 4. congo red 5. dehydrate 6. clear |
| Congo red results | Amyloid: deep pink to red Elastic tissue: pale pink muclei: blue |
| Crystal violet purpose | Demonstration of amyloid |
| Crystal violet fixative | 10% NBF |
| Crystal violet technique | 10-12 micrometers |
| Crystal violet control | Tissue containing amyloid |
| Crystal violet procedure | 1. Crystal violet 2. rinse 3. Mount |
| Crystal violet results | Amyloid: purplish violet other tissue elements: blue |
| Thioflavine T purpose | Demonstration of amyloid |
| Thioflavine T principle | Fluorescent dye that attaches to amyloid |
| Thioflavine T fixative | 10% NBF |
| Thioflavine T technique | 6-10 micrometers |
| Thioflavine T control | Section containing amyloid |
| Thioflavine T procedure | 1. Stain in Mayer heme 2. Wash 3. Stain with Thioflavin T 4. rinse 5. Differentiate in 1% acetic acid 6. wash 7. blot dry 8. mount |
| Thioflavine T results | Amyloid: Yellow to yellow green fluorescence |
Created by:
Ziek98
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