Hello all,

I'm performing brain immunolabeling in 15 days post-fertilization zebrafish larvae by using green and red channels (Alexa Fluor 488 and Alexa Fluor 594) with a 2-photon microscope. I noticed a lot of fluorescence coming from the blood vessels (especially in green channel, but I can also distinguish them in the red one), and found that this fluorescence was already present right after the fixation. I normally use 750nm as a wavelength, but I tried to image at different wavelength and I still saw this autofluorescence, in both channels.

As a fixative method, I'm using a mix of PFA / Glyoxal / PBS-Triton, but also tried with just PFA / PBS-T or PFA / PBS, and still saw this autofluorescence. I tried a 10min treatment with H2O2 to bleach the autofluorescence, but it didn't work (maybe the duration was not enough?).

Would you have any advice on how to remove this autofluorescence or how to manage it for image analysis?

Thank you for your help!