I'm not familiar with JB-4 in particular, but in general IHC is more dicey on resin-embedded tissues. I believe that it is the deplastination step that is quite harsh on the tissue and may cause loss of antigenicity. There are certain resins that are sold as "IHC-friendly", and may help for future projects, but of course not for your current tissues. Perhaps you could try a shorter deplastination step. Also, have you tried without any retrieval ? Have you tried a longer incubation of your primary ? Also, are you sure that you are supposed to have staining for your markers of interest in this tissue (ie. have you seen positive reactions for them on frozen or paraffin-embedded tissue equivalents) ?

  Maybe this paper can help: Immunohistochemical Identification of Lymphocyte Antigens Using a Modified Methacrylate Method Adapted for Standard Microtomes

Kenneth C.Bagley · Sayed F. Abdelwahab · Robert G.Tuskan · George K. Lewis · Frank J. Denaro

Thanks for the replies.

The main concern with JB-4 is that it cannot be deplastinated, so I've been playing with antigen retrieval steps. 

I know the marker works because I've done it on paraffin (looking for CD31).

My general primary incubation is always overnight, and from experience that's pretty much the best you can do.

Currently exploring the option of ABC detection method to greatly amplify the signal, so any suggestions would again be appreciated!

Thanks again

I see - i've looked at the specs and it's a GMA type of resin, which indeed can't be deplastinated, so I doubt that you'll have much luck with IHC. The manufacturers do not recommend it for IHC for that reason (the resin inhibits binding).