I was testing proper biotinylation of surface proteins using avidin-FITC staining on fixed cells (10min in 4% PFA).
FITC, which is expected to be only on the surface of the cells, were inside the cells and I was wondering if PFA will permeabilize the cells to internalize biotin and avidi-FITC.
Has anyone observe permeabilization of mammalian cells with 4% PFA?
Hi Sulgi, sorry to tell you, but dead cells are permeable.
You can fix them with military grade crosslinkers.
Dead cells are still permeable.
How can you tell whether your staining is intracellular or at the cell-surface? It might be helpful to post an image to show an example of what you think is intracellular staining.
My answer is very late. There is a problem on your biotinylation experiment. You might use aldehyde-conjugated biotin for cell surface biotinylation, but the most of primary amines on membrane proteins were already cross-liked each other by PFA. I suggest try biotinylation reaction at 4 degree (in live cells), stain with avidin-FITC (also 4 degree) and then fix the cells.
For your information, there are lots of biotin analogues in the cells. Good luck to you.
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