Serum Blocking: incubate sections in normal serum – species same as secondary antibody. Note: since this protocol uses avidin-biotin detection system, avidin/biotin block may be needed based on tissue type.
Primary Antibody: incubate sections in primary antibody at appropriate dilution in IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) for 1 hour at room temperature or overnight. Note: No serum blocking is needed if antibody diluent is used.
Secondary Antibody: incubate sections in Biotinylated secondary antibody in PBS for 30 minutes at room temperature.
Rinse in PBS-Tween 20 for 3x2 min.
Detection: incubate sections in ABC-Peroxidase Solution for 30 minutes at room temperature.
Chromagen/Substrate: incubate sections in peroxidase substrate solution
Counterstain with counterstain solution if desired.
Rinse in running tap water for 2-5 minutes.
Coverslip with mounting medium.