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Enzyme histochem

Staining

QuestionAnswer
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
Created by: CCF
 

 



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