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:
l 1% formalin in PBS for 10 minutes
l 80% methanol in PBS for 10 minutes
l Cold acetone for 5 minutes, air dry
l 4% paraformahyde
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 4°C. 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.