I would have thought that the rod outer segment can be washed away if not internalized. If the FITC adds some hydrophobicity that leads to increased membrane binding, you may try to add 0.5% BSA to your washing. For me, the washing seems the more important step, though the quenching with trypan blue is clever, but most likely not very efficient.

Dear Wissam,

with POS you mean photoreceptor outer sigment?

Did you measure in both FITC and a red channel? I guess through trypan blue, FITC-labeled POS expossed to TB shoul show FRET towards red channel (e.g. Cy3). At least, that is what happened in my experience - so maybe check in both green and red/orange red channel and see if you have signals from POS which are green only and some which are both green and orange/redish. Green only would be internalized at the moment you added TB, the other are exposed to TB.

Hope that helped

Best, Johannes

Adding Trypan blue (0.4%) to the sample just before visualization will help in minimizing toxicity issues while quenching surface fluorescence for you to quantify internalized FITC labelled POS. There is no need to wash the cells after adding TB. As you will be reading your sample straight way after adding TB, there won’t be any toxicity issue to your cells due of TB. (Add TB sample by sample, not to all sample (if you have many) at a time). TB also quenches the autofluorescence (if at all any by FITC)

Same can be done with FACS (if at all FACS based analysis is your choice). Advantage here is that TB fluoresces in our FL4 channel while FITC in FL1 channel. So, there won’t be any masking of FITC signal. If you got to any other dye for any other reason, make sure avoid those that fluoresces in FL4 channel as those can be masked by TB.

Hope it helps

Good luck