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HT/HTL: Chap13

Enzyme Histochemistry

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
Created by: Miellee



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