Sophie Tronel Please share you immunostaining protocol EXACTLY. You might be destroying tdTomato somewhere along the protocol.

I do an incubation with Methanol and H2O2 0.5% for 30 min, rince in PBS (4 X 10min) incubation in PBS-triton 0,3% + normal goat serum 5% and incubation in PBS-triton, NGS and anti-c-fos antibody 1/2000 (Cell Signaling) for 48h at 4°C.

Then incubation in goat anti rabbit A488 for 2 hrs.

Thanks

Sophie Tronel The problems I see here are

1) Fixation with methanol. Try fixation with PFA4% for 5-10 min. PFA is way better at keeping tdTomato safe.

2) Goat serum is too high, try 1%. The goal of incubating with NGS is to quench unnecessary fluorescence, thus you may be adding it too much.

3) In addition, the second incubation with Triton might be the problem. You only need to do permeabilization ONCE, no need to do it twice, remove second Triton completely.

4) Second incubation with NGS is also unnecessary and may produce this problem. Do the NGS incubation ONCE (in the step that you said "PBS-triton 0,3% + normal goat serum 5%").

Remove these excessive steps and see what happens.

thanks, I will try !