### Method: 1. 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 - Mount with coverslip