I usually stained my cells with antibodies and then performed the fixation with 4%PFA for 30 min but in my last experiment I performed the cell fixation first and then marked with my antibodies. I left my cells 12 hours in PFA1%, washed and ressuspended in my antibodies mix (CD3, CD4 and CD8). I performed this experiment twice and I observed that my CD3 staining did not work. I replaced my antibody and that did not work either.
I would like to know if someone has stained the cells after fixation? If so, do you know if CD3 staining works after the cell fixation?
Many antibodies do not stain fixed cells. Others do - just have to determine empirically. On the data sheet for the antibody, you can get a clue when they list applications. They often will say whether it works on fixed tissue sections or not. If it doesn't work on fixed cells, just stain first.
Hi,
PFA fixation should not affect CD3 staining. Did you stimulated your cells before staining? Some stimulations lead to CD3 internalization.
Here is the link to a BD list of anti-human antibodies with their compatibility with different fixation buffers.
https://www.bdbiosciences.com/documents/antibodies_human_cellsurface_marker.pdf
I agree with Gael that some stimulations (e.g. anti-CD3 or PMA/ionomycin) can induce CD3 internalization.
We always observe significant decrease of surface CD3 fluorescence intensity after stimulation.
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