It is fairly well documented in the literature that formaldehyde and glutaraldehyde fixation of cells causes the formation of large membrane blebs or blisters. These are quite unsightly artifacts that also interfere with the interpretation of my particular assay (which I won't go into). I therefore need to get rid of them, but I still need to fix the cells because I am looking at a time-dependent process that needs to be arrested by fixation. Here's a recent paper that actually looks into this bleb formation in a lot of detail:

http://rd.springer.com/article/10.1007/s00418-012-1058-5

That paper seems to suggest that fixation-induced blebs disappear at less than 2% FA, however I have gone down to 1% and still see them (even though they take slightly longer to develop). At such low concentrations I'm also not that sure that fixation is efficient, and I need to be certain the cells are fully fixed for biosafety reasons.

Here is my fixation protocol:

• HeLa cells in DMEM/10% FBS plated sparsely in 24-well plates

• Remove media, wash once with PBS 1X

• Replace with PBS/PFA of whatever concentration

• Incubate for 10 mins at room temperature in the biosafety cabinet

• Remove and wash once with PBS

• Replace with PBS and observe under bright field microscope

I see blebs develop almost immediately, and by 20 minutes almost every cell has at least a few small blebs, with many cells having lots of big ones. I use the PBS recipe from OpenWetWare and I buy 32% formaldehyde solution from Electron Microscopy Sciences which I dilute in the PBS.

Has anyone had to deal with this issue, and how did you resolve it? I have a couple of ideas that I'm going to try soon, but wanted to see if anyone had a quick fix (ha!). I'm going to try Dulbecco's PBS, or try using PBS/0.2% Triton X-100 (permeabilization buffer) after fixation instead of just PBS. Hopefully will either dissolve away all the blebbing membrane, or will prevent it from happening altogether.