- Proteins and Peptides
- Lysates and Cell Lines
1. Deparaffinize the section in 3 changes of xylene, 5 minutes each.
2. Wash the section in 3 changes of 96% 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 the citrate buffer, pH 6.0, 0.05% Tween-20*, and incubate at 95C in water bath for 20 minutes. (Alternatively adjust to your own protocol, keeping the required pH)
7. Remove the staining to room temperature and let the slide to cool (in citrate buffer, pH 6.0) for 20 minutes.
8. Rinse in distilled water.
9. Wash in 0.05 M Tris-HCl , pH 7.6 buffer supplemented with 0.2% of Tween-20 (buffer A) for 5 minutes.
10. Block the section with 5% skim milk in buffer A for 30 minutes. Drain blocking reagent and apply immediately the primary antibody.
11. For concentrated products: Incubate the section with primary antibody diluted in 0.05 M Tris-HCl, pH 7.6 buffer supplemented with 0.05% of Tween-20 at the dilution 1:100 - 200 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.
12. Wash twice 5 minutes with buffer A.
13. Apply the secondary antibody (the protocol depends on the supplier), and proceed to standard immunohistochemistry protocol (HRP - Peroxide - DAB).
14. Wash twice 5 minutes with buffer A.
15. Apply the chromogen (DAB), 10 minutes.
16. Rinse in water.
17. Stain in hematoxylin for 5 minutes.
18. Wash in water for 10 minutes.
19. Dehydrate the section in 2 changes of 96% benzyl alcohol for 5 minutes each.
20. Wash the section in 2 changes of xylene for 2 minutes each.
21. Mount the slide for observation.