Making Unpigmented Embryos with PTU

response to bionet.organisms.zebrafish 7/96
Query: I need to make zebrafish without eye pigment to check for expression of a gene in the eye. Does anyone have experience using PTU? I've worked with PTU enough to know it is pretty insoluble. I notice the Zeb Book says 0.003% PTU in Hanks. Do you just put in enought PTU to pretend it is 0.003% or is there some trick to getting it to go into solution? Any advice is welcome.
Thanks!
Dick Vogt
Responses:

Michael Brand: I make up a 0.3% stock which needs to be warmed to 65 degr for a while before it goes into solution - and I dilute it from there down to 0.003 %, and this works fine. The stock recristallizes with time, but goes into solution again upon reheating. I have not yet tried lower concentration stock solutions, but I suspect the ptu will still precipitate, just one does not see it , because I noticed that a 0.003% ready made ptu stock looses its pigment suppressing features after a while. I find prewarming the stock more convenient than having to weigh out this nasty stuff every time, and having to worry wether it will have gone into solution sufficiently well.

We have no eye pigment developing if we put the embryos into ptu early enough, that is, usually around the end of the day the embryos were layed, around the beginning of somitogenesis..

Ruth Bremiller: The protocol I have used is as follows:
Stock solution:
0.33 gm PTU per 100 ml fish water (this is a saturated solution and probably does contain crystals!) Add 1 ml of stock solution to 100 ml of fish water when the embryos are 10 to 20 hours old. I would guess, given the limited solubility of PTU, that trying to get it into solution in Hank's might be a problem.
Paul Meyers: We just make up a 0.3% solution, and squirt a pipette full into a beaker of embryos. It doesn't really go into solution -- it's a solution with a lot of white insoluble blobs floating in it -- but it is effective anyway.

However, for your purposes it may not do you much good. The eyes pigment up anyway, it's all the other pigment that gets supressed. It might slow the progress of pigmentation in the eye, but I don't know about that myself -- we tend to ignore that messy stuff up in front of the ears.

Anand Chandrasekhar: We make 0.4mM (0.006%) solution of PTU in fish water. It takes about 1-2 hr of vigorous stirring (longer the better) to dissolve completely. For treating embryos, I dilute the PTU water with an equal amount of fish water to reach the final concentration. PTU remains in solution and eye pigmentation is minimal or absent upto 72h.