We are using this antibody for IHC on 4% PFA fixed mouse lung sections to stain tdTomato and we have a lot of problems. We have tried a lot of protocols and different concentrations of the antibody. Now we are using the dilution1:400 in PBS + 0.05% Tween-20, which initially seemed the best one with the first lot, for 1 hour at room temperature, without antigen retrieval. Blocking is performed with 2% BSA for 20 minutes. The detection is done with the Mach 1 Universal HRP-Polymer for 30 minutes at room temperature. Sections are washed with PBS + 0.05% Tween-20 for 5 minutes. Color reaction is revealed by DAB. The problem is that the results on the same sections are not repeatable at least with this new lot number (GR 252580-5). Increasing the concentration of the antibody (1:100) we have a strong background and non-specific signals also in the negative control; decreasing (1:400) the amount, we lose the signal in the positive control... Any suggestions will be appreciated!!

Thank you

Michela