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Carbs & Amyloid

Staining of carbohydrates and Amyloid in the histology lab

QuestionAnswer
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
Created by: Ziek98