Immunohistochemical Elivison two-step method

1. Paraffin sections were placed in an oven at 67 ° C, baked for 2 hours, dewaxed to water, and rinsed three times with PBS pH 7.4 for 3 minutes (3 x 3').

2. Take a certain amount of pH=6.0 citrate buffer solution, add it to the microwave box, heat it to boiling in the microwave, place the dewaxed hydrated tissue section on the high temperature resistant plastic slice rack, and put it into the boiling buffer. After mid-range microwave treatment for 10 minutes, take out the microwave box and let the water flow naturally. Remove the slide from the buffer, rinse it twice with distilled water, then rinse 2×3' with PBS. (Note: not all antibodies require microwave repair, depending on the situation)

3. Add 1 drop of 3% H2O2 to each section and incubate for 10 minutes at room temperature to block the activity of endogenous peroxidase. Rinse 3 x 3' with PBS.

4. Remove the PBS solution and add 1 drop of the corresponding primary antibody (corresponding dilution factor) to each section and incubate for 2 hours at room temperature.

5, PBS rinse 3 × 5 '. The PBS solution was removed and 1 drop of polymer enhancer was added to each section and incubated for 20 minutes at room temperature. Rinse 3 x 3' with PBS.

6. Remove the PBS solution, add 1 drop of enzyme-labeled anti-mouse/rabbit polymer to each section, and incubate for 30 minutes at room temperature. Rinse 3 x 5' with PBS.

7. Remove the PBS solution and add 1 drop of freshly prepared DAB solution (diaminobenzidine) to each section and observe for 5 minutes under a microscope.

8, hematoxylin counterstaining, 0.1% HCl differentiation, tap water rinse, blue, sliced ​​by gradient alcohol dehydration, xylene transparent, neutral gum seal, dry and observe.

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