Hej,

has anybody experience with TSA in frozen sections and has also seen background/unspecific signal problems? I have used TSA from Perkin Elmer and ThermoFisher in FFPE sections and it always worked great. I am doing a lot of normal IHC stainings in frozen sections without any problems.

I use murine spleen, mLN, small intestine and human CRC tissue.

I tried acetone and formalin fixation without any difference.

I use a biotinylated antibody as a primary and then SA-HRP/SA-polyHRP (SuperBoost kit). If I use the SA-polyHRP from the ready to use bottle, the signal is all over the place even without any primary antibody. I diluted the SA-polyHRP even 1:300 but I am still getting background. Furthermore, the Tyramide alone (no prim and sec) gives also some background (although blocked 60min with 3% H2O2). I also tried different Tyramide incubation times but the signal/noise ratio is still not great. I really don't understand why this thing is behaving so strange in frozen sections. Anybody some recommendations or similar experiences?

Thanks!