I fixed my cells with 3.75% PFA at RT for 10min, washed them with FACS buffer, blocked them and finally stained them with CD80, CD64 and CD11b. I found CD80 expression is down compared to unfixed cells. Unfixed cells those went through the same procedure, just lack fixation, showed expected CD80 expression. Any idea why CD80 expression is lost in fixed cells? or should I try any other fixing solution?
Unfixed cells take-up fluorophore conjugated antibody by pinocytosis/endocytosis.
PFA crosslinks protein, and this can alter the structure of your target antigens. The anti-CD80 antibody you use may poorly recognize the antigen after fixation, thus reducing your FACS signal. Try doing your blocking and antibody labeling steps first, and then PFA-fix your cells, and see if that improves your signal. Incidentally, if you're blocking and doing staining steps at 4 degrees C, you should be able to mitigate any nonspecific staining due to Fc receptors or cellular uptake processes.
Thanks Stephen, but due to my experimental design, I can't stain the cells before fixing. Any idea then?
If the CD80 signal is down but not completely gone, that suggests to me that you still have some recognition of CD80 by the antibody. You might try doing a 2-step stain (anti-CD80 primary followed by fluor-conjugated secondary) to boost your signal, and/or reserving your brightest fluors (PE, APC) for this antigen.
I unfortunately don't have much experience with other fixation buffers (I use homemade 4% PFA in PBS) so I can't make any specific recommendations about that. If other labs near you use different fix buffers, and they're willing to provide you a few mL for an experiment, you could always see whether those solutions give you better results.
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