Are you including detergent in your fixation buffer (or in a subsequent step?) Detergent will cause pores to form in cell membranes even at relatively low concentrations as they allow hydrophobic lipid tails to solubilize. Many fixation procedures include detergent as pores are desirable for some antibody staining protocols.

I'm not including detergent in my fixation buffer..just using HBSS for first step in cleaning the cell surface..may i know further about the hydrophobiv lipid tails?

Hi a lot of fixatives will cause membrane pores/holes - especially the alcohol based ones (i.e. Methanol). Indeed if you fix with methanol then you do not need to perform a permeabilisation step (if you wanted to bind antibodies to intracellular epitopes for example).

If you want to avoid hole formation I would probably go with 4% paraformaldehyde (NOT formalin which is generally paraformaldehyde stabilised with ~15% w/v methanol).

Also don't use RNAlater (which is basically a mix of high concentration salts and can be considered to be a "precipitate" fixative as the alcohol fixatives are). This definitely causes membrane holes (I know as after RNAlater fixation all my Calcein dye leaked out of the cells!).

I'm not entirely sure of the mechanism of "hole" formation (I've never looked into it) but it is probably caused by a mechanical  "tearing" of the membrane during the fixation step, rather than lipid absorption etc.

Anyway, hope this helps,

Gary

Hi just want to add (for the record) it seems RNAlater does not seem to cause membrane holes - the lack of Calcein signal following RNAlater fixation may have been caused by the salts in RNAlater quenching the Calcein signal. I stained RNAlater cells with PI and there was only a few % positive cells.

Thanks,

Gary