Hi Yun,

Just a check when you look in the embryos into which you injected your cells do you still see these fluorescent dots in a similar location ?

The fact that these dots are visible in both the GFP and RFP channel makes it more likely that it is non specific fluorescence. Do you treat your fish with PTU to inhibit pigmentation ? You might think about this treatment at some point. If you overlay your brightfield with your fluorescence image do the dark spots correspond to darker patches in the brightfield ?

Even if you can't figure it out you might still be able to proceed with your experiment. After all your injected cells are presumable GFP+ but RFP negative in contrast to the other non specific patches which will be double positive.

Hope this somewhat helps.

Cheers,

Pieter

Hi Pieter Louwe and Elizabeth E LeClair

Thank you so much for those suggestions! I didn't realize PTU treatment was an option until I saw your replies and read more papers about it... My professor and I are also considering changing to another strain of fish, so I am wondering which one is better (I have read about casper, but I don't know if there's a strain better than casper)?

Thanks again!

Yun

Hi Yun,

You are welcome. In my experience breeding the casper line is quite a hassle so keep that in mind. A new line has been generated that you could look into, maybe they don't have the breeding issues Casper fish have, you could check with the authors.

Article A crystal-clear zebrafish for in vivo imaging

Maybe continue with PTU while you get a translucent line up and running and then run a couple of validation experiments in your translucent line.