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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 |
Congo red | Prolonged storage in 10% NBF will decreases staining intensity |
Congo red | 8-10 micrometers |
Congo red control | A tissue containing amyloid |
Congo red | 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 | Demonstration of amyloid |
Crystal violet | 10% NBF |
Crystal violet | 10-12 micrometers |
Crystal violet control | Tissue containing amyloid |
Crystal violet | 1. Crystal violet 2. rinse 3. Mount |
Crystal violet results | Amyloid: purplish violet other tissue elements: blue |
Thioflavine T | Demonstration of amyloid |
Thioflavine T | Fluorescent dye that attaches to amyloid |
Thioflavine T | 10% NBF |
Thioflavine T | 6-10 micrometers |
Thioflavine T control | Section containing amyloid |
Thioflavine T | 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 |