I accidentally used the same staining times for 2, 3 and 5 dpfs. 3dpfs look good after bleaching with H2O2. 2 and 5 dpfs (especially 2 dpfs) have a very black head, and even after 6 hours in bleaching solution, the head looks pretty dark. Do you think there's a way to overcome this?
Thanks!
The bleaching won't help much after the embryos are already stained. I've found that less is more when it comes to staining, as long as your embryos remain transparent. Have you tried using PTU (1-phenyl 2-thiourea) on the embryos to prevent pigmentation from developing? We add up to 0.003% PTU to our embryo media before 24 hpf, when pigmentation begins. If you keep the embryos in PTU they will remain transparent until you are ready to harvest them for your ISH and it will be easier to see the expression pattern with a shorter staining time.
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