To test for proliferative cells, I stained my tissue with Ki67- primary overnight and secondary 30min. This was followed by ABC 30min and DAB (1mL buffer + 1drop chromogen) for 30min. I counterstained with our regular haemotoxylin for 30sec. (Normally for other staining protocols I do 1-2 mins.) I used ammonia water as bluing agent and cleared in xylene.
All my slides seem to be false negative and are completely blue. I am not sure what in the protocol could produce that. I treated with H2O2 and ABC as usual. Any suggestions that could explain the results?
Dear Colleague,
Indeed, Haematoxylin counter-staining can mask your DAB signal.
Two suggestions:
1. ''Enhance'' your DAB by using a copper sulphate solution after DAB development (common practice). As a metal, this gives the DAB signal ''weight'' and allows one to discern positively stained cells fairly easily.
2. Alternately, you could use light green as your counter-stain. Quite useful when IHC localisation is nuclear.
Perhaps consider taking your slides back to water and differentiating the haematoxylin with acid/alcohol to ensure that you did have initial DAB signal.
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