I have stained mouse bonemarrow cells for flowcytometry with a cocktail of surface antibodies labelled with PE-Cy7 (CD45, clone 30-F11, TER119 and CD31, clone MEC13.3). I have then fixed the cells in PFA and stained them with an intracellular antibody.

My problem is that I loose some of the PE-Cy7 staining in the fixed cells. Aprox 10-20% more cells are PE-Cy7 negative in the fixed cells than in the unfixed cells.

I have read that CD31 antibody (clone MEC13.3) can show variable results in PFA fixed cells but the other clones should, as far as I understand, work also in fixed cells. I just didn´t expect that 10-20% would be CD31+?

Also, I thought it would be OK to stain the surface antigens first and then fix and stain the intracellular antigens. But maybe not….?

Can somebody help me with what is going on here?