I have been carrying out IHC to stain FLAG in fresh-frozen rat brain using FLAG M2 (F1804) with ABC then DAB, but am unable to identify positive staining, and seeing a high background.

I carried out controls (no primary/no primary no secondary) in which the no 1/2 control has very little staining but the no 1 control looks the same as the FLAG stain, so it seems that the background is very high from the secondary Ab.

I have tried increasing BSA in the blocking step, reducing the secondary concentration (goat anti-mouse), increasing the primary concentration, etc. I have also tried free floating IHC using PFA fixed tissue but see the same issue. I already include a H2O2 step, and have tried blocking endogenous biotin/avidin separately.

Does anyone know how to identify positive staining or reduce the background further?

Or should I move to Western blot or IF?