Enzyme Digestion and Microwave Heating Antigen Retrieval Method

COMBINED ENZYME DIGESTION AND MICROWAVE HEATING ANTIGEN RETRIEVAL METHOD 

Tissue specimens are fixed with 4% paraformaldehyde and embedded in paraffin at 60°C for 1 hr. Sections (4 pm thick) are mounted on gelatin-coated glass slides, deparaffinized, and rehydrated in distilled water. They are treated with 0.005% pepsin for 15 min at 37°C, followed by heating in 0.01 M citrate buffer (pH 6.0) in a microwave oven (300 W) at 80°C for 15 min. The sections are washed in distilled water for 5 min, rinsed in 0.01 M PBS (pH 7.2) for 15 min, and treated with 0.3-1% H202 to quench endogenous peroxidase activity. 

They are incubated in the primary antibody (PC10, diluted 1 200 in PBS containing 0.2% BSA for PCNA) for 1-2 hr at room temperature. The sections are washed five times for 5 min each with PBS and then incubated in the enzyme-conjugated secondary antibody in PBS containing 1% heat-inactivated normal rat serum for 1 hr. Horseradish peroxidase 

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