[Histonet] Fixation question - Cerebellar granular cells

[Guillermo Palchik]

Dear Histoneters,

I am doing some IHC on rat cerebellar granular cells (NOT TISSUE) and  
I need to fix them with PF.
I have gotten 4 different answers on how to go about this, and I  
wanted to run this by the list to see what you think.

1) Fix in COLD, 4% PF in 1x PBS

2) Fix in COLD, 4% PF in 1x PBS with 4% Sucrose.

3) Fix in WARM (37 C) ,in  4% PF in 1x PBS.

4) Fix in WARM (37 C), in 1x PBS with 4% Sucrose.

I get the fact that the PF might need to be at 37 C, since it is the  
temperature that the cells are in the incubator and it would probably  
temperature shock them. What about the sucrose? does it remove the  
water?

I'd appreciate your thoughts...
Best,

Guillermo

Guillermo Palchik
gp62 <@t> georgetown.edu