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HT/HTL: Chap13
Enzyme Histochemistry
Stain | Purpose | Principle | Fixative/Technique | Q.C. | Reagents/Procedure | Results | Notes |
---|---|---|---|---|---|---|---|
Acid Phosphatase | indicates inflammatory cells; muscle fibers in acid maltase deficiency also show increase in acid phosphatase; marker enzyme for lysosomes | phosphomonoesterases hydrolyze esters of orthophosphoric acid at pH4.0-6.0; alcohol residue of naphtol As-BI phosphate is reacted w/ hexapara to produce insoluble azo; simultaneous coupling diazonium into incubating med w/ substrate | Frozen at 10u | none; has internal control neutrophils and histiocytes will stain positive | PARAROSANILINE!!! incubate; rinse; methyl green; dehydrate to xylene | SITES OF ACID PHOSPHATASE ACTIVITY= Red; Background = green | Acid maltaes (type II glycogenosis) def will give positive rxn, lysosomal storage disease; make sure dehydration step is rapid; gall only |
Succinic Dehydrogenase (SDH) | further identify source of NADH diaphorase, only mitochondria show positive SDH | mitochondrial enzyme (oxidase) Kreb's, indix of activity of Kreb's; incubate w/ succinate in tetrazolium cmpd (NBT) hydrogen released = formazan | frozen at 10u | no other control | (NBT, physiological saline; succinate solution) Incubate at 37C; rinse in saline; fix in 10% formalin -saline; Rinse; Mount | Sides of SDH = BLUE | none |
Phosphorylase | muscle phosphorylase diagnosis of McArdle disease | produces amylose, length of which is proportional to amt of activity and when staine dw/ iodine vary in color depending on length; if branching, glycogen results; alcohol prevents action fo branching, glycogen added as primer and insulin as activator | None until ready to stain, cold acetone in 5min; Frozen at 10u | Sections of skeletal, excep in case of McArdle(no phosphorylase activity) | (Acetate buffer, phosphates, glycogen, PVP; Gram Iodine) Air dry; cold acetone; incubate 37C; shake off, was in 40%ROH; Air dry; abs ROH, air dry; stain dilute Gram iodine; mount in glycerine-iodine | PHOSPHORYLASE ACTIVE SITES = varying shades of brown, blue and purple; TOTAL ABSENCE OF ACTIVITY = Indicate sMcARdle disease | read slides immediately, fade rapidly; McArdle is one of 7 glycogen storage diseases, 4 of which involve muscle. |
a-Naphthyl Acetate ESTERASE | Non-specific; differentiates between TYPE II ATROPHY and NEUROGENIC ATROPHY as devnervated muscle fiberse stain dark, and type II do not; Motor end plates and lyosomes; histiocytes will stain, accumulations of Acetyl-Choline stains | hydrolyzed alcoholic residue wil couple w/ hexazotized prarosaniline solu. to give insoluble brightly colored azo-dye | Frozen section of skeletal muscle at 10u | section fskeletal muscle w/ inflammatory cells or motor end-plates | Incubate at 37C; wash; dehydrate to xylene Pararosaniline + Na-nitrite>>hexazotized parasosaniline; a-Naphtyle acetated + H2O>esterases>a-Naphthol + acetic; Naphthol + hexaz.pararos. --> azo dye! | Motor end plates = Brick red due to staining of Acetylcholine Esterase; Normal muscle fibers = Very pale yellow; Denervated muscle fibers = dark red-brown; Macrophagesand lysosomes = dark red-brown | Incubation medium will turn cloudy; wash slides well; TYPE I FIBERS STAIN SIGHTLY DARKER THAN TYPE II FIBERS |
Naphthol AS-D ESTERASE | identify GRANULOCYTES in the classification of leukemias or in chloromas, esterase is found int he lysosomes of some granulocytes | depending on their preferences for stustrates, specific or non-specific; SPECIFIC ESTERASE STAIN, can perform on paraffin sect; hydrolyzed alcoholic residue coup;es w/ hexpara give brightly colored azo-dye rxn | smeas: 2min aboslute methanol; tissue well fixed, EDTA ok, AIR DRY OVERNIGHT | Spleen for paraffin blocks | stain in fresh working esterase solution; wash, repeat if have to; counter in MAYER; was; rinse; blot, air-dry, xylene mount | POSITIVE CELLS (granulocytes and mast cells = bright red; Nuclei = blue; Background = unstained | make sure placed in xyelen after drying; only use glass |
ATPase | preincubation at various pHs will differentiate type I, type IIa and type IIb fibers (myopathic from neuropathic) CHECKERBOARD | muscle ATPase dependen ton pH allow for subtyping of fibers; Gomori-type metal precipitaion of hydrolyzed orthophosphoric acid w/ Ca; phosphate immedical combines w/ Ca2+ in solution; at alkaline pH, calcium phosphate is insoluble at sight of activity | FROZEN SECTIONS at 10u; three sides, 4.3, 4.6, 9.4 | no control needed | Barbital!!! CaCl; Cobalt-Cl; Ammonium sulfide (1) prepare barbital solutions at different pH's; incubate different times... | pH 9.4 = Type II fibers dark, type I fibers light to unstained; pH4.3 = Type I fibersdark, type II fibers light to unstained; pH4.6 = Type I fibers are dark, type IIA fibers light, type IIB fibers are intermediate | Ca2+ exchanged for cobalt; cobalt phosphate not visible so phosphate exchanged for sulfide!!!; ATPase activated by magnesium and inhibited by calcium --mitochondria; oppositite in muscle--> thinkabout how this works |
Alkaline Phosphatase | detection of regenerating muscle fibers | alkaline phosphatase hydrolze esters at basic pH, 8.6-8.8; naphthol reacts w/ fast red violet to produces insoluble dye; simultaneous coupling of diazonium cmpd incorporated into incubating medium | ROZEN SECTIONS at 10u | no other control necessary; alkaline phosphatase present in blood vessel walls | TRIS (trizma base); Paphthol As-Bi phosphate; N,N=dimethylformamide; incubate at room temp; rinse/ counter in hematoxylin (Mayer or harris) wash well; AQUEOUS MOUNTING | SITES OF ENZYME ACTIVITY = pink-red to red; Nuclei = blue | abnormal staining of perifascicular and endomysial connective in patients w/ inflammatory myopathy; regenerating fibers are selectively positive w/ this technique |
NADH Diaphorase | NAD reductase demonstrates abnormalities in mitochondria, Z-band material and sarcoplasmic reticulum | tetrazolium salt acts as hydrogen acceptor; NADH cannot reduce salt, attributed to flavoprotein enzymes thus DIAPHORASE (no carry( reaction NADH(reduced)-diaphorase-> NAD + H; H+ NBT --> formazan | Air-dried frozen at 10u | no other control | Incubate at room temp; rinse; mount w/ immumount of glycerine jelly (NBT solution; NADH);phosphate buffer) | SITES OF ENZYME ACTIVITY = dark purple deposits (Z-BAND MATERIAL, SARCOPLASMIC RETIC & MITOCHONDRIA strong); TYPE I muscle fibers = Dark purple; TYPE II MUSCLE FIBERS = Light | pH is critical and kept close to neutral (mitochondria can swell, enzyme solubility affected, NAD become unstable, basic (8.0) reduced coenqymes can reduce the salts; architectural changes in muscle: central cores, target fibers, nemaline, targetoid |