Hi Akin Sesver

I think 6% H2O2 may cause too much bubble formation in the embryos. You may try lower amount of H2O2 such as 1% or .05% for longer time to avoid bubble formation.

I also have a similar type of situation where I bleach mouse embryonic (E15-E18.5) skin with H2O2. If I use 5% H2O2, I also found air bubble trapped in skin. But if I use 0.1%, there is no bubble formation. Of course my tissues are very thin and you are using whole embryos. So considering these facts you may try 0.2-1% H2O2 treatment. Please let may know whether using lower H2O2 helps. Thank you.

This is a common problem, allegedly due to endogenous catalse activity in the tissue, or so I've been told. If your protocol allows it, you can try using H2O2 in methanol. It helps.

Hello Md Riaj Mahamud and Miloš Vittori

thank you both for your input!

I was able to try both conditions (1% H2O2 in PBST and 6% H2O2 in methanol)

and I am happy to say that I was succesful in having bubble-free but bleached embryos with the H2O2 in Methanol method! :)

The lower concentration of H2O2 still produced bubbles albeit slower.

Thanks!

Akin

Akin Sesver

I am glad that it helped. You may try using even lower concentration of H2O2 such as 0.2, 0.5%. Hopefully that'll generate even lower bubbles. Thank you!

That's great news!

All the best,

Milos

Hello

im struggling with the same problem here

bleaching step during Whole mount ISH lead bubbles inside of embryo and it cause membrane damage.

If it is okay with you, could you tell me which step did u add H2O2 + methanol?

Was it before rehydration step or after??

I would suggest before the rehydration step. It would make little sense to me to rehydrate the sample before incubating it in methanol.

I have the same problem even while putting the tissues in a vacuum for the bleaching step. I'm going to try the methanol approach while rehydrating in 50% MeOH and 1.5% H2O2 next.