Hi,

We have been doing this for many years for in situ hybridization. We use 
a 50 ml plastic tube filled with isopentane. This is placed in alcohol 
in a beaker. Alcohol is precooled with pieces of dry ice. The exact 
amounts needed are difficult to tell, we follow the freezing speed. We 
keep adding dry ice as it melts. Freezing of whole rat and mouse brains 
works fine. Do not leave the brains in isopentane too long, remove them 
from the tube with forceps as soon as they are frozen. Then keep them at 
-20--80 until cutting. Before cutting allow enough time in the cryostat 
to get the sample in appropriate cutting temp.

Feel free to write off the list if you need more details.

Best regards Pertti Panula


Guillermo Palchik wrote:
> Dear Confocalists,
> 
> Does anybody have a good working protocol for flash freezing fresh rat 
> brains (not perfused) ?
> I have been doing flash freezing by cooling Isopentane to -50 C and then 
> scooping the fresh brains into it and letting it sit for about 5-10 
> seconds, then scooping it into dry ice an letting it sit for a few 
> minutes and then into the -80 freezer.
> The problem is that I have many cracks (crystals) when I cut the brains 
> in the cryostat.
> After doing some research I found out that proper flash freezing is 
> supposed to prevent that...
> I have tried leaving it longer than 20-30 seconds in the Isopentane 
> (-50C) but the brains crack open.
> I also tried directly placing them into LN2 but it cracks after 5 
> seconds...
> I have heard that cooler Isopentane (-80 C) might work.
> Any ideas?
> Thanks
> Gil Palchik
> 


-- 
Pertti Panula
Professor, Research Director
Neuroscience Center
Institute of Biomedicine/Anatomy
POB 63
00014 University of Helsinki
Finland
Phone: +358 9 19125263
Fax: +358 9 191 25261
Mobile: +358 40 5922 323
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http://www.helsinki.fi/neurosci/panula.htm