Wash 1x in phosphate buffered saline (PBS)
Use Tris-buffered saline (TBS) instead of PBS for detecting phosphoproteins or when using alkaline phosphatase (AP) conjugated antibodies
Block non-specific binding sites
Wash 3x in PBS or TBS
Incubate with primary antibody
Wash 3x in PBS or TBS
(Optional) Incubate with secondary antibody
Wash 3x in PBS or TBS
(Optional) Incubate with amplification reagent
Wash 3x in PBS or TBS
Incubate with DAB or other substrate solution (for enzymatic labels only)
Wash in ddH2O
Counterstain
Dehydrate tissue sections (for organic mounting media only)