The size of the eggs are huge in comparison with some other insects and around 3.0-3.5 mm in lenght. So when I tried to stain with DAPI, I had autofluorescent in every part of the embryo and it is not penetrating in deeper parts of the embryo as can be seen on confocal. I am trying to see nuclei at different stages. Does anyone has any suggestion with different dye or technique?
I've used Hoechst 33342 successfully in whole mount of mammalian tissue. Not sure how penetration depth differs between Hoechst 33342 and DAPI, but I do know that Hoechst 33342 has better penetration depth than Hoechst 33258. Could be worth looking into/trying. Have you permeabilized your tissue with some detergent? I've also had success using 5% DMSO to help pull even antibodies into thick tissues.
Chelsea L. Fortin thank you very much for your answer. Maybe it can work and worth to try.. It seems like it has an additional ethyl group, thus, it's more permeable than other Hoechst and maybe than DAPI also (not sure). I use Tween-20 as detergent in PBT solution. DMSO, I never tried.. I remove chorion layer manually so I didn't use any chemical removal step for outer layers.
Insects are are bad about autofluorescence. You could try to use Propidium Iodine as a nuclear stain instead of DAPI and that would get you out of the blue wavelength range. Hoechst 33342 works like DAP adn binds to teh minor grove of DNA. You could also look up nuclear markers that are either in the far red or primary market that will allow you to label with a far red secondary to combat the endogenous autofluorescence. I would also block in PBST (1 x PBS with 1% BSA and 0.1% Triton X- 100) you could also boost the triton to 0.2% if the first concentration dies not work. Titon-X 100 is basically a detergent that will help with penetration. I hope that helps.
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