- Proteins and Peptides
- Lysates and Cell Lines
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 for 5 minutes.
6. Wash in 0.05 M Tris-HCl , pH 7.6 buffer supplemented with 0.2% of Tween-20 (buffer A) for 5 minutes.
7. For concentrated products: Incubate the section with primary antibody diluted in buffer A 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.
8. Wash twice 5 minutes with buffer A.
9. Apply the secondary antibody (the protocol depends on the supplier), and proceed to standard immunohistochemistry protocol (HRP - Peroxide - DAB).
10. Wash twice 5 minutes with buffer A.
11. Apply the chromogen (DAB), 10 minutes.
12. Wash in water for 10 minutes.
13. Stain in hematoxylin for 5 minutes.
14. Wash in water for 10 minutes.
15. Dehydrate the section in 2 changes of 96% benzyl alcohol for 5 minutes each.
16. Wash the section in 2 changes of xylene for 2 minutes each.
17. Mount the slide for observation.