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Carbs & Amyloid

Staining

QuestionAnswer
3 classifications of carbs Monosaccharides, oligosaccharides, polysaccharides
What is the only monosaccharide found in the body? Glycogen
Monosaccharides and oligosaccharides are_ Soluble in aq. solution, difficult to demo
4 groups of polysaccharides Group I: neutral polysaccharides Group II: acid mucopolysaccharides Group III: glycoproteins Group IV: glycolipids
Group I includes_ glycogen, starch, cellulose, chitin, PAS-positive
Group II includes_ Hyaluronic acid, heparin, chondroitin sulfates A, B, & C, human aorta, bovine cornea, "connective tissue mucins," PAS-negative
Group III includes_ ovimucoid, mucin, Paneth cell granules, sialomucins, sialogycoproteins, serum glycoproteins, "epithelial mucins," maybe PAS-positive
Group IV includes_ Cerebrosides: fatty subs. w/ carb, phosphatides: non-carb lipids, PAS positive
PAS purpose Demo polysaccharides, neutral mucosubstances, basement membranes
PAS facts 10% NBF or Bouin; blood smear - methanol, kidney 1-2 μm, other 4-5μm, QC: kidney; demo glycogen - liver or cervix
PAS solutions Periodic acid + Schiff Reagent + potassium metabisulfite + Harris w/ acetic acid or light green
PAS results Positive reaction: bright rose - nuclei: blue if hematoxylin - background: light green if counterstained
Schiff reagent solution sodium metabisulfite + HCL = sulfurous acid + basic fuchsin + activated charcoal + dH2O reacts w/ aldehydes to restore color structure
What substances give positive PAS reaction? Glycogen, neutral mucosubstances, some epithelial sulfomucins & sialomucins, colloid material of thyroid, pars intermedia of pituitary, basement membranes, fungal walls
PAS w/ diastase digestion purpose Demo glycogen
PAS w/ diastase digestion facts 10% NBF, formalin alcohol, or absolute alcohol QC: 2 sections of liver or cervix w/ glycogen - label one "w/," one "w/o"
PAS w/ diastase digestion solutions malt diastase solution + periodic acid + Schiff reagent + potassium metabisulfite + Harris w/ acetic acid
PAS w/ diastase digestion results Glycogen on "w/o" section: bright rose red - glycogen on "w/" section: no stain
Malt Diastase solution diastase of malt + phosphate buffer, pH 6
Phosphate buffer, pH6 NaCl + sodium phosphate + H2O
Mayer mucicarmine purpose Demo "epithelial" mucin
Mayer mucicarmine facts 10% NBF QC: small intestine, appendix, or colon
Mayer mucicarmine solutions Mucicarmine solution + Weigert hematoxylin + Metanil yellow or light green + H2O
Mayer mucicarmine results epithelial mucin, capsule of cryptococcus: deep rose to red - nuclei: black - other: blue or yellow
Mayer mucicarmine stock solution carmine + alum lake + aluminum hydroxide + anhy. aluminum chloride + 50% ethanol + 25% ethanol
Metanil yellow solution metanil yellow + acetic acid + H2O
Alcian blue, pH 2.5 purpose demo acid mucopolysaccharides
Alcian blue, pH 2.5 facts 10% NBF or Bouin QC: small intestine, appendix, or colon positive control
Alcian blue, pH 2.5 solutions acetic acid + alcian blue + nuclear-fast red
Alcian blue, pH 2.5 results acid mucopolysaccharides: dark blue - background/other: pink to red
Nuclear-fast red (Kernechtrot) solution nuclear-fast red + aluminum sulfate + H2O
Alcian blue, pH 1.0 purpose demo sulfated mucosubstances
Alcian blue, pH 1.0 facts 10% NBF or Bouin QC: small intestine, appendix, or colon positive control
Alcian blue, pH 1.0 solutions HCl + alcian blue, pH 1 + Nuclear-fast red
Alcian blue, pH 1.0 results sulfated mucosubstances: pale blue - background: pink to red
Alcian blue w/ hyaluronidase purpose Differentiate epithelial & connective tissue mucins, hyaluronidase digests out connective mucins
Alcian blue w/ hyaluronidase facts 10% NBF QC: 2 sections of umbilical cord - one labeled "w," one "w/o," may use small intestine, appendix, or colon as 2nd control for epithelial mucins
Alcian blue w/ hyaluronidase solutions hyaluronidase digestion soln. + acetic acid soln. + alcian blue + nuclear-fast red
Alcian blue w/ hyaluronidase results w/o, acid mucopolysaccharides, sialomucins: deep blue - w/, mucosubstances w/ hyaluronic acid, chondroitin sulfates A & C: marked loss of staining
Buffer solution, pH 6.0 potassium phosphate + sodium phosphate
Hyaluronidase digestion solution testicular hyaluronidase + buffer soln.
Alcian blue-PAS-hematoxylin purpose Differentiate between neutral & acidic mucosubstances
Alcian blue-PAS-hematoxylin facts 10% NBF, Zenker, kidney 2-3 μm, other 4-5μm QC: kidney or mucin control depending on diagnostic tissue
Alcian blue-PAS-hematoxylin solutions acetic acid soln. + alcian blue, pH 2.5 + periodic acid soln. + Schiff reagent + reducing rinse + Harris w/ acetic acid
Alcian blue-PAS-hematoxylin results acid mucosubstances: blue - neutral polysaccharides: magenta - substances stained by PAS & alcian blue: purple
Reducing rinse solution sodium metabisulfite + H2O prepare just before use
Müller-Mowry colloidal iron purpose Demo carboxylated & sulfated, (acid) mucopolysaccharides & glycoproteins, less specific than alcian blue, Prussian blue reaction
Müller-Mowry colloidal iron facts 10% NBF, Carnoy, alcoholic formalin; avoid chromate fixatives QC: small intestine, appendix, or colon
Müller-Mowry colloidal iron solutions acetic acid soln. + Müller-Mowry colloidal iron + ferrocyanide-HCl + Nuclear-fast red
Müller-Mowry colloidal iron results acid mucopolysaccharides & sialomucins: deep blue - nuclei: pink-red - cytoplasm: pink
Müller-Mowry colloidal iron working solution ferric chloride + acetic acid + H2O
Ferrocyanide-hydrochloric acid solution potassium ferrocyanide + HCl + H2O
What is amyloid? Fibrillar protein, deposits in tissue under pathologic conditions, contains some acid mucopolysaccharides
What is amyloidosis? Disease where deposits replace cellular elements causing progressive loss of function & eventual death
Primary amyloid Occurs in absence of disease, primarily muscle, heart, skin, tongue
Secondary amyloid Associated w/ disease, esp. inflammatory disease, e.g. RA & TB, primarily kidneys, liver, spleen, adrenal glands
Myeloma-associated amyloid Associated w/ diseases of immunological system, primarily muscle, heart, skin, tongue
Tumor-associated amyloid Associated w/ tumors, esp. those of amine precursor uptake & decarboxylation system
Alkaline Congo-red method facts Demo amyloid, alcohol or Carnoy 8-10 μm (or no birefringence) QC: sections w/ amyloid (brain, kidney) stain intensity decr. over time
Alkaline Congo-red method solutions Harris w/ acetic acid + alkaline salt soln. + Congo red working soln.
Alkaline Congo-red method results amyloid: deep pink to red - elastic tissue: pale pink - nuclei: blue
Alkaline salt solution 80% alcohol sat'd w/ NaCl + NaOH
Congo red working solution Congo red + 80% alcohol sat'd w/ NaCl + NaOH
Stock 80% alcohol sat'd w/ NaCl NaCl + 100% ethanol + H2O
Crystal violet purpose Rapid screening of amyloid, not as specific as Congo red
Crystal violet facts 10% NBF or alcohol 10-12 μm QC: section w/ amyloid polychromatic
Crystal violet solutions 95% sat'd w/ CV + Apathy mounting medium, seal w/ nail polish
Crystal violet results amyloid: purplish violet - other elements: blue
Modified Apathy mounting medium solution gum arabic + cane sugar + NaCl + thymol + H2O
Thioflavin T Fluorescent method facts Demo amyloid, less specific than Congo red 10% NBF 6-10 μm QC: section w/ amyloid
Thioflavin T Fluorescent method solutions Mayer + thioflavine T + acetic acid soln.
Thioflavin T Fluorescent method results amyloid: fluoresces yellow to yellow-green
How to test Schiff reagent quality? Schiff reagent should be clear/light amber; to test quality add formaldehyde, reddish-purple - good, deep blue-purple - solution breaking down
Best carmine facts Demo glycogen, less specific, alcohol, Bouin, Carnoy
Best carmine reagents carmine + K2CO3 + KCl NH4OH + methanol + ethanol + hematoxylin
Best carmine results glycogen: pink - nuclei: blue
Alcian blue is a: Copper phthalocyanin dye
Prussian blue reaction: Acidic potassium ferrocyanide, ferric iron reacts to form insoluble bright blue pigment
Congo red birefringence under polarized light should be: Bright green, 8-10 µm, too thin - red, too thick - yellow
Turnball blue reaction: Acidic potassium ferrocyanide, ferrous iron reacts to form insoluble bright blue pigment - ferrous ferricyanide
The colored product of the PAS reaction is formed by: Restoration of the quinoid structure
Created by: CCF
 

 



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