Immunoperoxidase staining of culture cells
1. Grow cells on sterile coverglasses or chamber slides overnight.
2. Rinse briefly with PBS
3. Fix cells by incubation with one of the following methods:
4. Rinse three times with PBS
5. Incubate with 1% H2O2 in PBS for 10 minutes to quench endogenous peroxidase activity
6. Rinse with PBS twice
7. Incubate slides for 30 minutes in 10% blocking serum in PBS (suppresses non-specific binding of IgG)
8. Blot excess blocking serum
9. Incubate with primary antibody for 1 hour at room temperature or overnight at 4กใC. Optimal antibody concentration is usually from 0.5-10ug/ml in TBS.
10. Wash three times with PBS
11. Incubate with biotin-conjugated secondary antibody for 30-45 minutes at room temperature. Again, optimal antibody concentration is usually from 0.5-10ug/ml in TBS
12. Wash three times with PBS
13. Incubate with streptavadin-HRP for 15 minutes. This step should be titrated as excess streptavadin-HRP can lead to high background
14. Wash with PBS three times
15. Incubate with freshly mixed DAB solution for 3-10 minutes. We highly suggest that you should observe the color developing under microscopy.
16. Rinse three times with distilled water
17. Counterstain with hematoxylin
18. Dehydrate with 70-100% alcohol and clear with xylene
19. Mount with coverslip
A. DakoCytomation Envision Doublestain System.