[Histonet] isopentane freezing of rat brain

[Charles Scouten]

Trying to slow down is all wrong.  Chill the isopentane to -80, as cold as you can get it with dry ice, and plunge instantly to the bottom, full immersion of the tissue in cold isopentane.  The poor appearance, especially in the middle, is freezing artifact due to freezing too slowly. 

The tissue will freeze vitreous, without expansion and resulting tissue damage, but gradually over time the ice will crystalize, even while stored at -40, and expand.  Thus, the sooner after freezing you can get to the sectioning, the better the tissue quality will be.


Cordially,
Charles W.  Scouten, Ph.D. 
myNeuroLab.com 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300  
FAX  314 522 0377 
cwscouten <@t> myneurolab.com 
http://www.myneurolab.com 


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Hector Gonzalez Pardo
Sent: Friday, February 11, 2005 10:51 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] isopentane freezing of rat brain

Hi,
I am using for the first time isopentane (2-methylbutane) to freeze my rat brains for histochemistry. However, when I do the histochemical staining, the tissue looks like "granulated" under the microscope, especially in the center. We usually immerse slowly (30 secs.) the entire brain on a weighing plastic boat in a glass vase containing isopentane chilled at -40 ºC. Then we wait for another 30 secs until it looks white on the surface, and we store it at -40ºC until sectioning using a cryostate. We used before a time-consuming procedure that worked, using cryoprotection with sucrose overnight, covering the whole brain with a cryogel (OCT compound / Tissue-Tek) and using a kind of freon-22 cooled by liquid nitrogen. Since we would like to continue using the easier isopentane procedure, does anyone know what are we doing wrong? Thanks! 
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