1 reply to this topic

[Danio ivanio]

Hey there,

Iam struggling with conditions of staining olfactory epithelia(Zebrafish). I did two attempts on fresh frozen tissue, dissecting in cold PBS then embedding in tissue tec followed by cryosectioning. When I perform the immunostaining I usually fix in cold Aceton for 15 Min, then wash the tissue and proceed to 1st AB without Antigen retrieval. My washing solutions have 0.1% Triton. When I mount the tissue and counterstain to observe it I always have kind of a mashy morphology. I also tried with Tween20 instead of Triton, still no change.

I know that fresh frozen tissue is more delicate and that nice morphology is hard to achieve. So what do you think about fixing in PFA for few minutes instead of Aceton? Will it help? I still want to ommit AR...Should I leave out the detergend at all?

Has anybody experience with epithelia or similar and can give few suggestions?


Thanks
Ivan

[bob1]

You could try fixing in PFA and then mounting in wax and doing your IHC from there rather than cryosectioning, which is probably where the loss of morphology is happening.