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Enzyme Histochemistr
Enzyme histochemistry in the histology lab
| Question | Answer |
|---|---|
| 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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