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Enzyme histochem
Staining
| Question | Answer |
|---|---|
| Muscle fiber cells: | 30-80 µm diameter, up to 35 cm long, multiple elongated nuclei, cross-striations, actin & myosin |
| Type I fibers | "Slow-twitch," red muscle fibers, predominantly aerobic, good blood supply, much mitochondria, lipids, myoglobin |
| Type II fibers | "Fast-twitch," white muscle fibers, anaerobic, high resistance to fatigue few mitochondria, myoglobin, poor blood supply, rich in glycogen, glycolytic enzymes |
| 3 types of type II fibers | Based on ATPase reaction: IIA, IIB, IIC, IIC are undifferentiated fibers not normally seen in adult muscle |
| Pathological changes to muscle fibers include: | Atrophy, hypertrophy, changes in location, appearance of nuclei, presence of inflammatory cells, degenerating/regenerating fibers, incr. in connective tissue |
| Oxidation | Addition of oxygen, loss of H, loss of e- |
| Reduction | Loss of oxygen, gain of H, gain of e- |
| Enzymes & pH | Most best neutral, near pH 7, if prefer acid pH won't work in alkaline & vice-versa |
| Enzymes & fixation | Most are removed or destroyed by fixation, some sensitive to freezing & thawing |
| Enzymes & temperature | Best at optimal temperatures, esp. simultaneous coupling, most activity destroyed above 50°C |
| Enzymes & substrate | Very specific as to substrate, enzyme unchanged by reaction, sometimes can't use optimal substrate concentration b/c poor solubility or hydrolyzes more slowly |
| Enzymes & inhibitors | Excess diazonium salts, fixatives, heat, some metallic ions may decr. or completely stop enzyme activity |
| Preservation of enzymes | Best fixed, or fixed before freezing, b/c diffusion artifact in unfixed frozens, 3-4°C formalin, calcium formalin, or acetone, store 30% sucrose soln. w/ 1% gum acacia @ 4°C for weeks |
| Blood smear & muscle biopsy preservation | Smear- fix once dry, muscle biopsies - frozen, post-fix if possible |
| Best method of freezing for enzymes | Isopentane in liquid nitrogen |
| Hydrolases | Enzymes that act on substrate usu. thru addition of water, 3 subclasses - esterases, phosphatases, peptidases |
| 4 type of rxn of hydrolases: | Simultaneous capture/coupling, post-incubation coupling, self-colored substrate, intramolecular rearrangement |
| Simultaneous capture/coupling | Most common technique, as rxn product is released it's captured by diazonium salt (gives insol. azo dye) or metallic ion |
| Post-incubation coupling | Depends on formation of insol. rxn prod. that remains at rxn. site for entire incubation period |
| Self-colored substrate | Rxn product insol. & colored, no coupler agent req'd |
| Intramolecular rearrangement | Substrate is sol. & hydrolysis causes molecular rearrangement resulting in insol. colored prod. |
| Esterases | Break bond between carboxylic acid & alcohols, phenol, or napthols, "specific" & "nonspecific" esterases, most can hydrolyze α-napthyl acetate, diazonium coupling agent, fastest coupling at pH 6.0-6.5 |
| Phosphatases | Break bond between alcohol & phosphate group, lots in plant, animal tissue 2 kinds - alkaline phosphatases & acid phosphatases, make visible w/ variety of techniques, usu. Gomori-type metal precipitation, form colored metallic sulfide |
| Lyases | Enzymes that add chemical groups to double bonds, don't stain for |
| Isomerases | Enzymes that rearrange chemical group on substrate, don't stain for |
| Ligases | Enzymes that combine two substrates, don't stain for |
| Oxidoreductases | Include oxidases, peroxidases, & deydrogenases, |
| Oxidases | Utilize molecular oxygen as H+ acceptor, forms water |
| Peroxidases | Catalyze ox. of substrates by H2O2 |
| Dehydrogenases | Remove H+ from substrate & transfer to coenzyme, thru e- transport system to oxygen, forms water |
| Diaphorases | Dehydrogenases that only ox. NADH or NADPH coenzymes, flavoproteins - contain flavine adenine dinucleotide |
| Transferases | Enzymes that transfer fxnal group from one compound to another |
| Phosphorylase | Transferase that moves phosphate groups, lots in plant, animal tissue, catalyze many reversible rxns, in vivo - degradation of glycogen |
| Freezing muscle biopsy specimens | Orient so cross-section, use isopentane in liquid nitrogen, stain ASAP, may store in freezer in closed container for several days, may see some reduction in enzyme activity |
| α-Napthyl Acetate Esterase stain for muscle biopsies purpose | Differentiate between type II atrophy & neurogenic atrophy, demos motor end plates & lysosomes in inflammatory cells |
| α-Napthyl Acetate Esterase stain for muscle biopsies facts | Sections: frozen, unfixed 10 µm, QC: skeletal muscle w/ inflammatory cells or motor end plates |
| α-Napthyl Acetate Esterase stain for muscle biopsies incubation solution | 0.2M phosphate buffer, pH 4.7 pararosaniline stock α-napthyl acetate in acetone sodium nitrite 4% pH 6.8-7.2 w/ 1N NaOH or 1N HCl |
| α-Napthyl Acetate Esterase stain for muscle biopsies results | normal muscle fibers - v. pale yellow motor end plates - brick red (b/c acetylcholine esterase) denervated muscle fibers, macrophages, lysosomes - dark red-brown azo dye end product |
| Pararosaniline stock | Pararosaniline dH2O conc'd HCl |
| Napthol AS-D Chloroacetate Esterase technique purpose | ID granulocytes as leukemias or chloromas b/c esterase in some lysosomes |
| Napthol AS-D Chloroacetate Esterase technique facts | Fixation: smears - 2 min. abs. methanol/formaldehyde wash w/ dH2O, tissue - any fix, (only EDTA-decal'd bone), paraffin sections air-dried overnight, QC: most aspirate or buffy coat smears have internal control, normal aspirate, spleen for paraffin |
| Napthol AS-D Chloroacetate Esterase technique reagents | Soln. I: esterase solutions A, B, & C pH 6.3 w/ 0.1N HCl or 0.1M sodium barbital Soln. II: Napthol AS-D chloroacetate w/ N,N-dimethylformamide, I+II Mayer hematoxylin |
| Napthol AS-D Chloroacetate Esterase technique results | positive cells (granulocytes & mast cells) - bright red nuclei - blue background - unstained |
| Esterase solution A | ρ-Rosaniline conc. HCl dH2O |
| Esterase solution B | sodium nitrite dH2O |
| Esterase solution C | 0.1N HCl 0.1M sodium barbital |
| ATPase stain purpose | Differentiate type I, type IIA, & type IIB fibers, determine myopathic from neuropathic processes |
| ATPase stain facts | Sections: frozen, unfixed 10 µm, QC: skeletal muscle has internal control only reliable stain for fiber typing |
| ATPase stain reagents | barbital acetate buffer sodium barbital incubating solution (sodium barbital in CaCl w/ dH2O) CaCl 1% CoCl 2% ammonium sulfide |
| ATPase stain results | pH 4.3 - type II fibers light to unstained, type I dark pH 4.6 - type IIA fibers light, type IIB fibers intermediate, type I dark pH 9.4 - type I fibers light to unstained, type II dark Cobalt sulfide visible |
| Barbital acetate buffer working | sodium acetate x 3H2O sodium barbital, dH2O HCl, water pH 4.6 w/ 1N NaOH or 0.1N HCl |
| Acid Phosphatase in muscle biopsies purpose | Indicate inflammatory cells, muscle fibers in acid maltase deficiency show incr. in acid phosphatase |
| Acid Phosphatase in muscle biopsies facts | Sections: frozen, unfixed 10 µm, QC: skeletal muscle has internal control - neutrophils & histiocytes stain positive |
| Acid Phosphatase in muscle biopsies reagents | Incubating medium: veronal acetate buffer napthol AS-BI phosphoric acid N,N-dimethylformamide pararosaniline HCl sodium nitrite 4% dH2O pH 4.7-5 w/ 0.1N NaOH Methyl green w/ veronal acetate buffer & pH 5.0 w/ HCL > 4.0 w/ acetic acid |
| Acid Phosphatase in muscle biopsies results | sites of acid phosphatase activity - red background - green alcoholic residue of napthol AS-BI phosphate visible, azo dye end product |
| Veronal acetate buffer | sodium acetate x 3H2O sodium barbiturate dH2O |
| Alkaline phosphatase stain for muscle biopsies purpose | Detection of regenerating fibers |
| Alkaline phosphatase stain for muscle biopsies facts | Sections: frozen, unfixed 10 µm, QC: skeletal muscle has internal control - alkaline phosphatase present in blood vessel walls |
| Alkaline phosphatase stain for muscle biopsies reagents | Incubating medium: TRIS buffer (TRIS, dH2O, HCl) napthol AS-BI phosphate N,N-dimethylformamide dH2O fast red violet Mayer or Harris hematoxylin |
| Alkaline phosphatase stain for muscle biopsies results | sites of enzyme activity - pink-red to red nuclei - blue azo dye end product |
| NADH Diaphorase purpose | Demo abnormalities in mitochondria, Z-band materials, & sarcoplasmic reticulum |
| NADH Diaphorase facts | Sections: air-dried frozen, 10 µm QC: skeletal muscle has internal control |
| NADH Diaphorase reagents | incubating medium: NADH NBT soln. saline soln. (NaCl, dH2O) phosphate buffer, pH 7.4 dH2O pH below 8.0 mount w/ Immu-mount or glycerine jelly |
| NADH Diaphorase results | sites of enzyme activity (Z-band, sarcoplasmic reticulum, mitochondria) - dark purple deposits type I muscle fibers - dark purple type II muscle fibers - light formazan - final reaction product |
| Succinic Dehydrogenase (SDH) purpose | ID source of NADH diaphorase activity - only mitochondria show SDH activity |
| Succinic Dehydrogenase facts | Sections: frozen, unfixed 10 µm QC: skeletal muscle has internal control |
| Succinic Dehydrogenase reagents | Incubating medium: NBT solution phosphate buffer, 0.2M sodium succinate solution dH2O physiological saline 10% formalin-saline solution |
| Succinic Dehydrogenase results | Sites of SDH activity (mitochondria) - blue |
| Phosphorylase stain for muscle purpose | Diagnosis of McArdle disease, glycogen storage disease, which is caused by a single enzyme defect |
| Phosphorylase stain for muscle facts | Sections: frozen, unfixed 10 µm, just before staining fix 5 min cold acetone QC: skeletal muscle has internal control, if McArdle suspected use control w/ known phosphorylase activity |
| Phosphorylase stain for muscle reagents | Incubating solution: acetate buffer, pH 5.9 (acetic acid, sodium acetate) α-D-glucose-1-phosphate adenosine-5-phosphate sodium fluoride polyvinyl pyrrolidone (PVP) insulin glycogen Gram iodine mount in glycerine-iodine |
| Phosphorylase stain for muscle results | phosphorylase activity - shades of brown, blue, & purple no activity - indicates McArdle disease, read immediately, fade rapidly, can re-stain w/ dilute iodine |
| How to prevent granular artifact in enzyme stained slides that cannot be dehydrated? | Air-dry & dip in xylene before mounting coverglass |
| NBT | Nitro blue tetrazolium, soluble, chromogenic, substrate of alkaline phosphatase, H+ acceptor for diaphorase & NAD-dependent dehydrogenase |
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CCF
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