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Enzyme Histochemistr

Enzyme histochemistry in the histology lab

Skeletal Muscle Presence of elongated nuclei at regular intervals. Regular intervals of cross-striations.
Type I fibers Slow twitch
Type II fibers Fast twitch
Type II fibers high resistance to fatigue
Enzymes Proteins that catalyze chemical reactions in biologic systems by temporarily combining with their specific substrate or the compound on which they act.
Catalyst Changes the rate of the reaction
Cofactors Can be metal ions or complex organic molecules called coenzymes.
Oxidation Addition of oxygen Loss of hydrogen loss of electrons
Reduction Loss of oxygen Gain of hydrogen Gain of electrons
Most enzymes are demonstrated best when... At a pH of 7.0 (There are exceptions)
Preservation of enzymes is done best with Cold formalin or cold calcium formalin
Preferred method for freezing muscle Isopentane and liquid nitrogen
Isopentane and liquid nitrogen should reach this temp before adding muscle to freeze -150 degrees Celsius
Hydrolases Esterases Phosphatases Peptidases
Hydrolases reactions 1. Simultaneous capture or coupling (Most common) 2. Post incubation coupling 3. Self colored substrate 4. Intramolecular rearrangement
Esterases Enyzmes capable of breaking the bond between carboxylic acid and alcohols, phenol, or naphthols
Phosphotases Hydrolytic enzymes that break the bond between an alcohol and a phosphatase group
Oxireductases 1. Oxidases 2. Peroxidases 3. Dehydrogenases
Oxidases Uses molecular oxygen as the hydrogen acceptor wight he resulting formation of water
Peroxidases Catalyze the oxidation of substrates by hydrogen peroxide
Dehydrogenases Removes hydrogen atoms from an organic substtrate
Transferases Enzymes that transfer a functional group from 1 compound to another
Phosphorylases Transferases that transfer phosphate groups
Alpha-napathyl esterase Useful for differentiating between type II atrophy and and neurogenic atrophy.
Alpha-napathyl esterase Hydrolyzing
Alpha-napathyl esterase Motor end plates: Brick red due to staining of acetylcholine esterase Normal muscle fibers: Very pale yellow Denervated muscle: Dark red-brown Macrophages and lysosomes: Dark red brown
Napathol AS-D chloroacetate esterase technique To identify granules in the classification of leukemias or in chloromas
Napathol AS-D chloroacetate esterase technique Hydrolyzing
Napathol AS-D chloroacetate esterase technique Buffy coat smears have an internal control.
Napathol AS-D chloroacetate esterase technique 1. Esterase 2. wash 3. counterstain with Mayer heme 4. wash 5. Blot and air dry then mount
Napathol AS-D chloroacetate esterase technique Positive cells (Granulocytes & mast cells): bright red Nuclei: Blue Background: Unstained
Napathol AS-D chloroacetate esterase technique Only glass should be used
ATPase Differentiate between type I, type IIA, and type IIB fibers. Helps separate myopathic from neuropathic processes
ATPase Dependent on pH. Hydrolyzing
ATPase No fixative
ATPase 10 micrometers
ATPase internal control
ATPase pH 9.4: Type II fibers are dark and type I fibers are light to unstained pH 4.3: Type I fibers are dark and type II are light to unstained pH 4.6: Type I fibers are dar and type IIA are light, and type IIB are intermediate
ATPase Stain will fade
Acid phosphatase Presence of inflammatory cells in the biopsy
Acid phosphatase Hydrolyzing
Acid phosphatase No fixative
Acid phosphatase 10 micrometers
Acid phosphatase Internal control present
Acid phosphatase 1. Incubate 2. rinse 3. methyl green counter
Acid phosphatase Acid phophatase activity: Red Background: Green
Acid phosphatase Only glass should be used
Alkaline phosphatase Detection of regenerating muscle fibers
Alkaline phosphatase hydrolyzing
Alkaline phosphatase no fixative
Alkaline phosphatase 10 micrometers
Alkaline phosphatase built in control
Alkaline phosphatase 1. Incubate 2. rinse 3. counterstain in heme
Alkaline phosphatase Sites of enzyme activity: Pink-red to red Nuclei: Blue
NADH diaphorase Abnormalities in mitochondria, Z-band material, and sarcoplasmic reticulum
NADH diaphorase Dehydrogenase
NADH diaphorase No fixative
NADH diaphorase 10 micrometers
NADH diaphorase Internal control
NADH diaphorase 1. Incubate 2. rinse 3. mount
NADH diaphorase Sites of enzyme activity: Dark purple deposits (Z-band material, sarcoplasmic reticulum, and mitochondria all react strongly) Type I muscle fibers: Dark purple Type II muscle fibers: Light
NADH diaphorase pH must be kept close to neutral for optimal results
Succinic dehydrogenase Further identify the source of NADH diaphorase activity, because only mitochondria show positive SDH activity.
Succinic dehydrogenase Oxidase
Succinic dehydrogenase Frozen
Succinic dehydrogenase 10 micrometers
Succinic dehydrogenase internal control
Succinic dehydrogenase 1. Incubate 2. Rinse 3. 10% Formalin saline 4. Rinse 5. Mount
Succinic dehydrogenase Sites of SDH: blue
Phosphorylase Diagnosis of McArdle disease
Phosphorylase Phosphorylases
Phosphorylase Cold acetone
Phosphorylase 10 micrometers
Phosphorylase 1. Air dry 2. Cold acetone 3. incubate 4. shake off excess and wash in 40% alcohol 5. fix in absolute 6. stain in gram iodine 7. mount
Phosphorylase Phosphorylase activity: Brown, blue and purple Absence of activity: McArdle disease
Phosphorylase Fades rapidly
Created by: Ziek98



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