[Histonet] cryostat brain in situ / tecan

[Susan Renn]

Hello

I am looking for a good protocol for DIG labeled in situ on brain cryostat
sections (from a fish).  I am using a tecan liquid handler.  The protocol
that I have right now has a dehydration step which I am not familiar with
from my previous work with in situ hyb in drosophila.... Is this step
necessary for brain sections? What does it do?

 

 

Also, I have seen protocols which call for fixation before sectioning and
others that call for quick freezing in oct and fixation after sectioning.
What are the pros and cons of these two techniques.  Furhtermore, if one
does fix after sectioning, should it be done immediately after sectioning,
after the sections have warm dried for 20 min, or just before hybridization
after the sections have been stored at -80 for some time?

 

Any answers are greatly appreciated.

 

Suzy