Deparaffinization

1.  Incubate slide at 60℃ for 60 minutes.

2.  Deparaffinize in Xylene for 10 minutes and repeat one more times.

3.  Hydrate in 100% alcohol for 5 minutes, in 95% alcohol for 5 minutes, in 85% alcohol for 5 minutes, in 75% alcohol for 5 minutes.

4.  Dip into Distill Water for 5 minutes.

5.  Dip into PBS (pH 7.6), leave for 5 minutes, and repeat two times.

Antigen Retrieval

6.   Bring 500 - 2000 ml 50 mM EDTA buffer solution(pH7.0) to the boil in a stainless steel pressure cooker.

7.   Put the slide into staining rack and lower into pressure cooker ensuring the slide is well immersed in EDTAbuffer solution.

8.   When the pressure indicator valve has risen after 3-4 minutes, incubate for 1-2 minutes.

9.   Cool the slide naturally to room temperature.

10.Dip into distilled water, leave for 5 minutes, and repeat two times.

11.Dip the slide in PBS for 5 minutesand repeat two times.

12.Immerse slides in 3% H₂O₂ (in fresh methanol) for 15 minutes at room temperature.

13.Wash with distilled water two times, 5 minutes each time.

14.Wash withPBS (pH 7.6) two times, 5 minutes each time.

Staining with Primary Antibody

15.Dilute primary antibody with 3% BSA in PBS. Cover the tissue section on the slide with diluted primary antibody (use 50 – 150μl for each slide).

16.Incubate at 37℃ for 30 minutes or at room temperature for 60 minutes (The optimal incubation time, incubation temperature, and antibody dilution should be determined by the individual laboratory).

17.Wash with PBS two times, 5 minutes each time.

Staining with Secondary Antibody

18.Incubate with 100-200μl Polymer Enhancer.Incubate 30 minutes at 37℃.

19.Wash with PBS for 3 times, 5 minutes each time.

20.Incubate with 100-200μlPolymerized HRP and incubate 30 minutes at 37℃

21.Wash with PBS for 3 times, 5 minutes each time.

23.Wash with distilled water for 2 times, 5 minutes each time.22.Add DAB solution and incubate 3-10 minutes(The reaction progress and the optimal time should be determine according to microscope).

24.Counterstain sections in hematoxylin if required,wash with distilled water.Immerse slides in 0.1% HCl- ethanolfor 1-10 seconds, wash with distilled water.

25.Dehydrate through 95% ethanol for 1 minute, 100% ethanol for 2×3min,Xylene for 2×3min,and coverslip with mounting medium.