dapi? You mean DAP, the chromogen substance? In my protocol, I add DAP after applying secondary antibody because it is a marker to see whether there is bond between antigen and primary antibody (monoclonal), which can be detect by bond between that primary antibody and secondary antibody (polyclonal). Thus I think, the secondary antibody must be placed before the chromogen. The principle of IHC or ICC is similar to ELISA’s.