It is difficult to really determine what is going wrong without a more detailed protocol. For instance, after I let my tissues (kidney) sit in 4% PFA for 2 hr at RT after perfusing the rat, I rinse the tissues 3 x 10 min in PBS. Then I wash the tissue for 30 min in 100 mM Tris-HCl followed by 3 x 10 min washes in PBS. I place my tissue in 30% sucrose overnight and then embed the tissue the following day in OCT in cryomolds. After cutting the blocks and placing the sections on slides, I wash 3 x 5 min in TBS. This is followed by either 0.2% triton or 1% SDS in TBS for 10 min. This is followed by 3 x 5 min washes in TBS with 0.02% tween. Then I block in 1% milk in TBST for 1 hr at RT followed by antibody incubation.

What detergent are you using for your permeabilization step?

Hi Naz

Have you tried sodium citrate buffer (pH 6.0) in boiling water bath for 10 min. for antigen retrieval?

Hi Brandon, Thanks for your answer. Does the Tris-HCl do the antigen retrieval? I use 1% TritonX-100 for 20 mins for permeabilization! I have tried 1% SDS for antigen retrieval but it didn't work. :(

Hi Meetu,

Yes I have tried sodium citrate but that didn't work and really mangled the sections :(

hey Naz,

The 100 mM tris HCl is not for antigen retrieval. It is necessary to incubate your samples with it (or something like it) to inactivate the paraformaldehyde. I read that you have tried the sodium citrate buffer and was surprised that that did not work for your antigen retrieval.

Naz Rasiwala Please look at the supplemental data in this paper. It contains a comprehensive protocol for IF: https://www.pnas.org/content/117/20/10876.abstract