I am attempting to use the counter stain in fish between 15-25 days post fertilization, and the dye appears to penetrate, but the patterns I am seeing under confocal do not match the organ I am expecting to see. Could this be an issue with dye pentrance due to the size of the embryo? And would it be feasible to leave the fish in the dye for significantly longer periods of time (overnight for example) to increase penetrance and make the staining more cohesive?
Thanks!
Hi Rachel! I don't see a problem to stain a fish at 25 days post fertilisation. The only problem could be the pentrance. But do you want to perform the staining with the fish alive or dead? Because if they are dead you can increase the time of the permeabilisation step and increase the concentration of the detergent that you use. You can also leave over-night of course but be careful of the over-staining!! And if you are applying when still alive be careful that to much/ or long incubation of dye will not kill your fishes. Maybe start increasing incubation periods by one hour and you will see (annoying yes...).
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