Hi

I'm having a problem with fixation step in cell prep for confocal microscopy.

I used 6% PFA for fixation before.
Recently my collegue suggested to use glutaraldehyde for fixation since it gives clear staining for unstable membranes, so I mixed both methods for fixation [glutaraldehyde fixation plus PFA fixation]

Though the latter really gave clearer staining for membranes compared with the former, there's false positive binding with our negative control Ab.

Does anybody ever have this problem?
Or any suggestions?

Thanks!

we just use 10% formalin and it doesn't give us to much bdg. You might try that.

Did you have a problem with your membranes that you switched away from PFA? I also use PFA and have no problems but I am not doing complicated confocal, just basic stuff.

PFA fixation was fine for my expriments, but it was just that GA + PFA fixation may give cool images for apoptotic cells. [all those clear images for apoptotic blebs!!!] Maybe GA fixation gives more nonspecific binding in this case...
Anyway, I'm back to PFA fixation though I'll do some more control exp...

Thanks anyway!!

Are you sure it isn't autofluorescence instead of non-specific binding? Aldehyde based fixatives are famous for giving really bad "autofluorescence" and this needs to be quenched before staining. GA is worse than PFA for this.