I am currently working on a project that involves photo-activation of fluorophore containing probes to spatiotemporally tag the surface of cultured cells or fixed tissue. There are two photo-activated probes we are working with. One has an azide species and the other is a primary amine. The tagging works well with live cells but has a very high background with fixed cells. To try to overcome this, I tried to quench the PFA Schiff base moieties with 0.1% sodium borohydride, 2M glycine, or 1M pH 8 Tris. The Tris works relatively well with a 1 minute quenching procedure for the azide reactive probe species, but the other quenching agents haven't worked at all. I have not been able to reduce the background for the primary amine reactive species with any quenching agent yet. Next I plan to try to reduce and alkylate the cell surface with DTT and iodoacetamide after an initial PFA quenching procedure. Has anyone experienced this reactive nature post PFA quenching and are there any suggestions on how to successfully reduce the surface so that the photo-reactive specifies won't readily couple with cells before UV activation?