Grow cells on sterile coverglasses or chamber slides overnight.
Rinse briefly with PBS
Fix cells by incubation with one of the following methods:
1% formalin in PBS for 10 minutes
80% methanol in PBS for 10 minutes
Cold acetone for 5 minutes, air dry
4% paraformahyde
Rinse three times with PBS
Incubate with 1% H2O2 in PBS for 10 minutes to quench endogenous peroxidase activity
Rinse with PBS twice
Incubate slides for 30 minutes in 10% blocking serum in PBS (suppresses non-specific binding of IgG)
Blot excess blocking serum
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.
Wash three times with PBS
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
Wash three times with PBS
Incubate with streptavadin-HRP for 15 minutes. This step should be titrated as excess streptavadin-HRP can lead to high background
Wash with PBS three times
Incubate with freshly mixed DAB solution for 3-10 minutes. We highly suggest that you should observe the color developing under microscopy.
Rinse three times with distilled water
Counterstain with hematoxylin
Dehydrate with 70-100% alcohol and clear with xylene