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You can also try a heat-denaturation step after the first secondary. I think, someone reported in this forum, that it worked.Heating in Retrievalbuffer, 75°C, 5-10 min. This should denaturate your first primary and you get a "brand new" slide for the second part of your doublestain. The question is, if the fluorochrom will survive.This methods works well with Peroxidase+DAB / Alkalic Phosphatase+Red on tissueslides.Your cells are fixed, so they could withstand the heat.Gula
Look at this staining procedure as two separate sequential IF stainings. Shouldn't matter that you are using two mouse primaries IF you get the blocking right....