I would like to stain surface, intracellular cytokines, and nuclear transcription factors on the same samples if at all possible. I have been cautioned that the aggressive detergents needed to perm for nuclear staining might make it impossible to retain the fluorophores in the cytoplasm on cytokines...? Which kit would I use, the nuclear (foxP3) or the normal ICS (fix/perm)? Any advice is greatly appreciated. Thanks!
HI Ebony,
It is possible to stain the transcription factors and intracellular cytokines together. However, for intracellular cytokines staining, it is recommended to stimulate the cells with PMA and Ionomycin in presence of brefeldin A. After stimulation you can fix the cells by 1% paraformaldehyde for 15 min at 4 degree Celsius. Permeabilize the cells with detergent (preferably saponin) or you can use perm/fix solution available with BD Biosciences or Biolegend. After permibilizing, stain the cells with all antibodies together. They will bind specifically to the target antigen. Preferably use fluorochrome conjugated antibodies.
Hope this may help you
It is possible to do this but the fixative and permeabilisation agent that you use will depend on the transcription factor you are staining for. Tbet, Eomes, FoxP3, Bcl6 need a buffer like the eBioscience FoxP3 buffer or a BD kit.
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