[Histonet] Please help me with my cryosections

[Yuqing Zhang]

Hi everybody!

A friend recommended this forum to me as he said lots of experts here could help
me with my horrible cryosections.

Right now, I am working on a gene expression pattern in zebrafish retina. I need
to section the eye before I see the expression clearly. So I do the whole mount
in situ hybridization first, then embed the embryos in TFM (some thing like OCT)
for cryosections. But I have several problems with my sections.
 
1. Some sections have big holes inside, but the cell shape is still fine
, this kind of situation happens more in younger embryos (36
hour post fertilization)
2. In some sections, the cells seemed became one big block, and there are some
strange patches inside the section, cells seemed aggregated to each
other
3. Some sections have small holes, the cell shape is totally indistinguishable.


I have tried several approaches to find out the reason, but non of them worked.
1. Slides of different brand.
2. gradient sucrose concentration (10%, 20%, 30%). or only 15% sucrose.
3. Do not fix embryos in PFA after in situ, keep them in PBST.
4. Freeze sample in liquid nitrogen. Or freeze them in cryostat.

I wanted to attach the pictures and protocol I am using, but the system said it
it too big to be posted. 

Thanks a lot in advance for you help.

Yuqing Zhang