Hi all,
I stained tdTomato+ cells (PBMCs and Jurkats) with eBioscience Foxp3 / Transcription Factor Staining Buffer Set for the foxp3 and I completely lost the signal of tdTomato. I tried to fix them first with 1% PFA (10min RT) and then permeabilize, which didn't work, and if I only fix them with PFA I can see the signal so I'm assuming that something on the kit is interfering with the detection of tdTomato.
I was wondering if anyone had the same problem and if yes any ideas?
Permeabilization buffers such as TritonX100 or Tween-20 usually denature the fluorescent proteins such as TdTomato or RFP, GFP. As Urs Mörbe suggested if you want to combine TdTomato with antibody staining you need to use an anti-TdTomato antibody to restore the signal as antibodies readily bind to denature and no longer fluorescent TdTomato.
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