you can try blocking with 10 % BSA prior incubation with primary antibodies..in my case, things improved. This article might be of help to you

http://www.ncbi.nlm.nih.gov/pubmed/22548649

Question, can you define your high autofluorescence? Is the tissue itself autofluorescent, meaning it wasn't properly stored and the formalin polymerized? Or, is the fluorescence coming from your assay?

The tissue itself is autofluorescent. This is a inherent property of FFPE tissues and has been the topic of a number of papers:

http://www.ncbi.nlm.nih.gov/pubmed/17548270

http://www.ncbi.nlm.nih.gov/pubmed/24722432

http://www.ncbi.nlm.nih.gov/pubmed/21370006

I have tried the photobleaching and Sudan Black protocols as partially described in the literature and do not have routine access to a confocal microscope. What I am asking for is a specific, step-wise protocol that someone has used for successful immunofluorescence on FFPE tissues, despite the inherent autofluoresecence of FFPE.

There are two options, one is to try Maxblock autofluorescence reducing agent from Dianova and the other one is to try heating paraffin at 80C for 30 minutes followed by an immediate transfer into xylene...Hope this helps!!!

Thank you Sandhya! It is already part of our regular routine to heat at 60C for 30-40 minutes followed by an immediate transfer to xylene (within 1-2 seconds), but the autofluorescence is terrible still. We haven't yet purchased a reagent tailored for autofluorescence quenching, but I think that is probably the next step.