I am working with PFA fixed mouse muscle sections. Although I have no trouble visualising my protein meaning that the protocol works quite well, the high background is killing me So this is the deal, I use goat anti-mouse to block the endogenous mouse IgG prior to using primary mouse monoclonal antibody. The secondaries are biotin conjugated Goat Anti Mouse and streptavidin conjugated alexafluor. Now I have tried using both 4% BSA and 10% Normal Goat Serum (NGS) as blocking reagents, however the bkg is not reduced. It looks like the muscle has some endogenous biotin which could be causing the bkg, so I used an avidin-biotin blocking kit but to no avail. I block with the avidin solution for 10 min, wash for 3x5' with PBS, then block it with biotin solution for 10', wash for 3x5' with PBS and then apply the primary antibody. I am running out of answers here, so if any of you have experienced this kind of a problem before or have experience with the avidin-biotin blocking kit, please do leave some suggestions

I don't know whether biotin is the problem here. How about conjugate your 1st Ab with dye, so that you can avoid all these steps?

Hmm, then it should not be due to biotin. The problem is likely due to fixation - blocking - washing. If you are fixing with PFA, take care to wash carefully. It can cause bad background. I would suggest some quenching with, e.g. NH4Cl, for a few times. Also, check out the thread of nkm about immunofluorescence background (it is in this forum, 1st page). There are lots of useful suggestions.