I wonder if possible to fix your cells first prior to both, membrane antigen and IC staining. All protocols available suggest to stain for membrane markers, fix, permeabilize and finally stain for intracellular antigens. I need to get some time between harvesting cells and staining procedure and I hoped fixing cells could help in order to prevent any changes that might occur on the surface or in cytoplasm. Please advice if I am wrong. Thank you!
You can fix the cells first prior to staining for membrane markers but you run the risk that the fixitive (typically 4% paraformaldehyde) will denature the epitope of the membrane marker and your antibody will not bind and you will get a possible false negative result.
After harvesting the cells you can try putting the cells on ice which should slow them down and prevent changes from occuring.
Good Luck!
for my opinion it is good to use 4% glutaraldehyde. paraformaldehyde is very quickly polimerized and form formic acid
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Both Drs. Masi and Zonovkin are correct in their statements. There are some limitations to prefixing before extracellular staining, and every protein/antibody is different, so you won't know until you try. Paraformaldehyde (4%), gluteraldehyde (1%) and methanol are all commonly used fixatives, each with pluses and minuses. But epitope blocking, see Dr. Masi's note, is a consideration if you get negative results, but is typically not a problem. In short, you should try. I would start with 4% paraformaldehyde fixation, then a triton/saponin permeabilization, then a BSA block, then your IC stain.
Dr. Daniel Lee Adams give you the best variant, but I think that it will be good to clarify that solution of gluteraldehyde must be on PSB(7,34) and better to use saponin, becuse triton have destructive effect for membranes for some tissues.
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