IHC-P protocol

1. Deparaffinize the section in 3 changes of xylene, 5 minutes each.
2. Wash the section in 96%, 80% and 70% benzyl alcohol for 5 minutes each.
3. Rinse in distilled water.
4. Block the endogenous peroxidase by incubating the tissue in 3% hydrogen peroxide (H2O2) for 10 minutes.
5. Wash in distilled water.
6. For antigen retrieval: immerse the slide in Tris-EDTA buffer, pH 9.0, 0.05% Tween- 20*, and incubate at 95˚C in water bath for 30 minutes.
7. Remove the staining to room temperature and let the slide to cool for 15 minutes.
8. Rinse in distilled water.
9. Wash in 0.05 M Tris-HCl , pH 7.6 buffer supplemented with 1% of Tween-20 (buffer A) for 5 minutes.
10. For concentrated products:Incubate the section with primary antibody diluted in buffer A at the dilution 1:100 - 1:300 for 1 hour in the closed wet chamber.
    For RTU products: Incubate the section with primary antibody (ready to use) for 1 hour in a closed wet chamber.
11. Wash twice 5 minutes with buffer A.
12. Apply the secondary antibody (the protocol depends on the supplier), and proceed to standard immunohistochemistry protocol (HRP – Peroxide – DAB).
13. Wash twice 5 minutes with buffer A.
14. Apply the chromogen (DAB), 10 minutes.
15. Wash in water – 10 minutes.
16. Stain in hematoxylin for 5 minutes.
17. Wash in water – 10 minutes.
18. Dehydrate the section in 2 changes of 96% benzyl alcohol for 5 minutes each.
19. Wash the section in 2 changes of xylene for 2 minutes each.
20. Mount the slide for observation.