"As extracellular staining requires primary antibody binding before fixation". This is not clear at all, why can you not simply fix the eggs and then use the antiserum.

I agree with Jan's point above! You should be able to aldehyde-fix your samples without permeabilizing too much.

If fixation messes with the antibody staining, my first suspect would actually be epitope/antigen masking, not loss of membrane integrity!

Hi @Jan Adrianus Veenstra and Alberto Sepulveda Rodriguez, I need to perform the primary antibody step before the fix because I need to capture protein which is in extracellular matrix. If I fix and perform washes after that, it washes off all the extra cellular protein. I tried the regular fix followed by primary antibody incubation protocol which resulted in no detection of extra cellular but only the intracellular protein. Please let me know if am conceptually wrong here but others have successfully done it (however that was in larvae and not in adult fly tissue).

I see. Sorry!

My only other thought is that you could try to do your primary antibody incubation step in different conditions that will better support the health of your cells. Depending on the specifics of your culture and of your ihc target, you could even try staining in the cells' own media. If that's unfeasible, maybe just try some form of minimal media instead of just a buffered saline-based antibody dilution buffer.

Hi Thank you for the input. I am trying to do washes with minimal buffer as you suggested (without salt) and playing with the incubation times. Will keep you posted.