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HT/HTL: Chap7

Carbohydrates and Amyloids

Special StainPurposePrincipleFixative/TechniqueQ.C.Reagents/ProcedureResultsNotes
Alcian Blue-PAS-H differentiate between neutral and acidic mucosubstances acidic mucosubstances are stained with alcian blue; neutral mucosubstances are stained by PAS 10%NBF or Zenker's; paraffin of routine 4-5u, kidney 2-3u kidney or a mucin control, depends on diagnostic tissue 3% Acetic acid; Alcian blue; wash; 0.5% P.A.; wash; Schiff; reduce(metabisulfite); wash; stain in Harris w/ acetic acid;wash; dehydrate Exculsively acid mucosubstances = blue; neutral polysaccharides= magenta; certain substances will be colored by both PAS and Alcian blue = purple none
Muller-Mowry Colloidal Iron demonstrate CARBOXYLATED & SULFATED MUCOPOLYSACCHARIDES, GLYCOPROTEINS at low pH, colloidal ferric(Fe3+) ions are absorbed by carboxylated and sulfated mucosubstances; excess washed out and classic prussian blue occurs 10% NBF, Carnoy, or alcoholic formalin preferred; NO CHROMATE FIXATIVES; paraffin at 4-5u section of small bowel, appendix of colon (GI TRACT after stomach!) Rinse in 12% acetic; stain in workin gcolloidal iron solution; rinse in acetic; immerse in ferrocyanide-HCl acid sol; wash; coutner w/ nuclear fast red; wash *can microwave* ACID MUCOPOLYSACCHARIDES AND SIALO MUCINS = deep blue; NUCLEI = pink-red; CYTOPLASM = pink strongly acidic mucins won't stain (like alcian blue); PAS can be counter; more intense than alcian blue, but not specific; can use digestion; CRYPTOCOCCUS NEOFORMANS STAINS WELL; can see background staining make sure FRESH
Alkaline Congo Red demonstarte AMYLOID in tissue green birefringence is considered most specific; congo red is benzidine and can react w/ cellulose; treating w/ alkali aids in release of internal H-bonds releasing dye binding sites; congo red linear and binds to azo and amine groups Alcohol or Carnoy preferred; 10%NBF, Bouin or Zenker ok; can't be long in 10% formalin decrease in stain intensity! PARAFFIN 8-10u needed for birefringence sections/ amyloid; do not keep many controls as staining intensity will decrease; SMALL NEWLY FORMED DEPOSITS STAIN BETTER THAN OLD BIG ONES Stain in Harris hematoxylin w/ acetic acid; wash; place in alkaline salt solution; stain in working Congo Red solution; dehydrate to xylene AMYLOID= deep pink to red; Elastic Tissue = pale pink; Nuclei = blue; BRIGHT APPLE GREEN BIREFRINGENCE SHOWS IN POLARIZED LIGHT specific to amyloid! can get false positives due to excess dye retained n tissue; sodium chloride and high alcohol depress dye ionizationa nd acid=base type staining = clean bkground;thickness of section is CRITICAL
Crystal Violet RAPID screening method of Amyloid NOT AS SPECIFIC either "metachromatic" due to mucopolysaccharide or basic dyes of crystal violet and methyl violet selectively reacts w/ amyloid, polychromatic; acid in staining solution prevents overstaining of cytoplasm 10% NBF or Alcohol; paraffin at 10-12u sections w/ amyloid Stain in working Crystal Violet; check control slide; rinse; mount AMYLOID= purplish violet; other tissue elements= blue careful of "bleeding" of aniline dyes during mounting; can air dry sections; can stain overnight in crystal violet
Thioflavine T Fluorescent good method for AMYLOID not as specific as Congo Red method w/ polarization Thioflavine T fluorescent dye that attaches to amyloid and requires no differentiation; background is black due to aluminum hematoxylin 10%NBF; 6-10u section w/ amyloid must be used Stain in Mayer hematoxylin; wash; stain in filtered thioflavine T; rinse; differentiate in acetic acid; wash; mount w/ fluorescent AMYLOID - fluoresces yellow to yellow-green pH1.4 (acid) increases selectivity of dye; LIPID GRANULES, JUXTAGLOMERULAR GRANULES AND MAST CELLS may fluoresce yellow, but is differentiated easily
PAS Reaction demonstration of polysaccharides, NEUTRAL mucosubstances and basement membranes olidation of 1,2 glycol to aldehyde by PERIODIC ACID/PERIODATE; Schiff reagent (pararosaniline/basic fuchsin + sulfurous acid)-reduction-causes masking of chromophores=LEUCOFUCHSIN; Schiff reaction = restores color. Metabisulfite used to remove excess 10%NBF or Bouin; Blood in Methanl; 4-5u paraffin (kidney 1-2u) Section of KIDNEY most sensitive; if for glycogen use liver or cervix (with both endo and exo) Periodic Acid; wash; Schiff warm to room temp;Wash w/ metabisulfite(optional); running tap water; counter w/ Harris hematoxylin w/ acetic acid; wash to blue; run through GLYCOGEN, NEUTRAL MUCOSUBSTANCES, EPITHELIAL SULFOMUCINS AND SIALOMUCINS, COLLOID MATERIAL OF THYROID AN DPARS INTERMEDIA OF PITUITARY, BASEMENT MEMBRANES AND FUNGAL WALLS = positive PAS (bright rose) can use fast green
PAS-Diastase demonstration of glycogen in tissue sections sensitive method for glycogen; diastase and a-amylase act on glycogen to break then down and washed out of section; PAS rxn is then used 10%NBF, formalin alcohol, absolute alcohol two control sections of LIVER on w/ and one w/o; can also use CERVIX incubate "with" at 37C diastase; wash; "w/" "w/o" in periodate; wash; Schiff; metabisulfite; wash in tap; counter with Harris in acetic; wash; dehydrate to xylene GLYCOGEN= bright rose on w/o and nil from w/ Malt diastase may loosen sections; can also use human silava; NO PICRIC ACID fixatives(resistant to digestion); CERVIX is special case (w// digestion stratified squamous of ecto will be negative)
Mayer Mucicarmine staining of "EPITHELIAL" MUCIN EMPIRICAL stain; Aluminum is believed to form a chelation complex w/ carmine = net positive charge attaching to acid groups of mucin 10%NBF; 4-5u paraffin section of small intestine, colon, or appendix Stain in WEIGERT hematox; wash; stain in MUCICARMINE; rinse remove excess H2O; METANIL; dehydrate to xylene MUCIN= Deep rose to red; CAPSULE OF CRYPTOCOCCUS = deep rose to red; NUCLEI = black; Other tissue elements = BLUE or YELLOW careful of overstaining w/ metanil or hematox; USEFUL FOR CRYPTOCOCCUS NEOFORMANS; but maybe not so important elsewhere
Alcian Blue, pH2.5 demonstration of ACID mucopolysaccharides Alcian blue = copper phthalocyanin basi dye water soluble and colored blue; in 3% acetic acid (ph2.5) stains both sulfated and carboxylated acid mucopolysaccharides and sialomucins (glycoproteins); by forming salg linkages w/ the acid groups of the mucin 10%NBF or Bouin; 4-5U section of small intestin, appendix, colon = positive control 3% acetic acid; ALCIAN BLUE; rinse in 3% acetic acid to remove excess; wash in running; rinse; counter w/ NUCLEAR FAST RED; wash; dehydrate to xylene WEAKLY ACIDIC SULFATED MUCOSUBSTANCES, HYALURONIC ACID AND SIALOMUCINS = dark blue; background = pink to red make sure Alcian Blue certified by Biological Stain Commission is used
Alcian Blue, pH1.0 demonstration of SULFATED mucosubstances in 0,1N HCl acid solution pH1.0, only sulfated acid mucopolysaccharides and sulfated sialomucins (glycoproteins) are stained 10%NBF or Bouin; 4-5u section of small intestine, appendix or colon positive control 0.1N Hydrochloric acid; 1% ALCIAN BLUE in HCl acid; rinse in HCl acid to remove excess; blot dry, avoid H2O; counter w/ NUCLEAR FAST RED; wash in DI-water; dehydrate to xylene SULFATED MUCOSUBSTANCES = pale blue; BACKGROUND= pink to red none
Alcian Blue w/ Hyaluronidase differentiate Epithelial and connective tissue mucins staining will disappear or be reduced when tissue sections containing HYALURONIC ACID, CHONDROITIN SULFATE A, or CHONDROITIN SULFATE C are digested w/ hyaluronidase; glycoproteins remain unaffected 10%NBF preferred two sections of umbilical cord (w/ & w/o); small bowel, appendix or colon can be used to demonstrate epithelial mucins (preheat each reagent to 37C) each section and w/ control in digestion, w/o in buffer; wash in running; 3% acetic acid; ALCIAN BLUE; wash running; rinse; counter w/ NUCLEAR FAST RED; wash running; dehydrate to xylene W/O DIGESTION, ACID MUCOPOLYSACCHARIDES AND SIALOMUCINS = deep blue; W/ DIGESTION, MUCOSUBSTANCES CONTAINING HYALURONIC ACID AND CHONDROITIN SULFATES A&C = marked loss of staining none
Created by: Miellee



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