ENZYME HISTOCHEMISTRY.................pptx

Enzyme histochemistry

1. Enzyme histochemistry

2. ENZYMES - DEFINITION Enzymes are biological catalysts that accelerates a chemical reaction. ENZYME HISTOCHEMISTRY Utilising histochemical techniques to demonstrate enzyme in tissues.

3. Preservation For good demonstration, maximum amount of enzyme activity should be preserved - with accurate localisation and by preventing diffusion of enzymes. Lyo -enzymes: dissolved in cytoplasm; likely to diffuse Desmo -enzymes: attached to cytoplasmic constituents; less likely to diffuse

4. SAMPLE PREPARATION FOR ENZYME HISTOCHEMISTRY Fresh preparation or frozen/cryostat/freeze dried preparations are necessary. Autopsy tissue: unsuitable for demonstration of enzymes. Cold fixative should be used to preserve maximum enzyme activity Eg : Ice cold formalin, ice cold alcohol

5. Factors affecting Enzyme Activity Temperature : optimal temperature in EHC is 4°C -30 °C PH optimum pH-7 ALP-9.2 ACP-5.0

6. Inhibitors Specific Inhibitors :eserine for choline esterase Non-Specific Inhibitors: heat Competitive Inhibitors: malonate ions for succinate dehydrogenase Activators :Mg ions for ALP

7. 2 TERMS USED IN ENZYME TECHNIQUE Primary Reaction Product : the product of the reaction of an enzyme on a substrate Final Reaction Product: t he product of an insoluble uncolored PRP, which has been made colored or opaque

8. METHODS OF DEMONSTRATION 1. Simultaneous coupling
2. Post Incubation coupling
3. Self- Coloured substrate
4. Intra-molecular Rearrangement
5. Metal Precipitation

9. Simultaneous coupling Single incubation Mixture: Substrate + Diazonium salts

➤ Eg : Azo dye method for Phosphatase

10. Post Incubation Coupling Substrate (for enzyme hydrolysis) and Diazonium salts (Coupling) are in separate solutions

11. Advantages: Optimal pH for enzyme substrate reaction and possibly different pH for coupling can be achieved. Deleterious effect of diazonium salts on enzyme substrate reaction can be avoided.

12. Self Colored Substrate

13. Intramolecular Rearrangement

14. Metal Precipitation A type of Simultaneous Coupling but uses Metallic lons instead of Diazonium salts.

15. Use of Controls Substrates, Diazonium salts and other components deteriorate with time leading to false positives and false negative reactions. Therefore, Control sections should be carried together with test sections. Positive Controls: to demonstrate that all reagents are working Negative Controls: prepared by a) destroying the enzyme in the test section by immersing in boiling water for 15 minutes b) by including a specific enzyme inhibitors c) By omitting the substrate from the incubation medium

16. APPLICATION OF ENZYME HISTOCHEMISTRY PATHOLOGICAL DIAGNOSIS OF MuSCULAR DISORDERS 1. 5' Nucleotidase 2. Adenosine Tri-Phosphatase 3. Cytochrome oxidase 4. Lactate adehydrogenase 5. Choline Esterase 6. Mono-amine-oxidase 7. Phosphorylase

17. DIAGNOSIS OF HEMATO-LYMPHOID MALIGNANCIES 1. Peroxidase 2. Alkaline phosphatase 3. Acid phoshphatase 4. β- glucuronidase 5. Non-specific esterase 6. DOPA-Oxidase 7. Chloroacetate -esterase

18. Determination of Alkaline Phosphatases Optimal activity at 9-9.6 pH
Activated by Mg, Mn and Co ions
Cyanide and Cysteine inhibits ALP- ase activity- thus, incorporated in incubation medium as a control METHODS 1. Gomori’s Calcium Phosphate method (Metal Substitution)
2. Azo Dye Coupling methods – Simultaneous Coupling – Post Coupling

19. AZO-DYE SIMULTANEOUS COUPLING METHOD USING SUBSTITUED NAPHTHOL

20. Incubation mixture: 1. Tris Buffer pH 9.1
2. Distilled Water
3. Substrate Solution ( Naphthol phosphoric acid)
4. Fast Garnet GBC salt

21. Procedure : Fix sections in methanol or cold acetone in freezing temperature for 30 sec.
Rinse in Distilled water.
Keep the section in the incubation mixture for 1-2 hrs at RT.
Rinse in water.
Counter stain with hematoxylin
Mount in glycerine .

22. RESULT Positive cells – granular dark brown

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