[Histonet] Cryoprotection Issues with Unfixed Mouse Hearts

[gayle callis]

Adam, 

It is not a good to sucrose cryoprotect fresh tissues.  Only well fixed
tissues should be cryoprotected to lessen freezing artifact caused by large
water ice crystals.  Charles Scouten, on his website discusses this, and if
one cryoprotects fresh tissues, you change the osmolarity within
tissues/cells and the tissues shrivel up.  This is probably the
damage/changes you are observing. Tissues that are well fixed with NBF or a
PFA mixture should be sucrose cryoprotected. 

For fresh tissues, dissect heart from mouse, embed and snap freeze with a
proper RAPIC snap freezing method. If you would like, I will privately send
a simple snap freezing that eliminates use of precooled isopentane. Avoid
freezing in a cryostat or a freezer, even -80C as freezing will be too slow
and at warmer temperture than true/rapidsnap freezing methods. This will
also cause freezing artifact in both fresh or sucrose cryoprotected fixed
tissues.  

Be sure to read Dr. Scouten's comments on snap freezing and its effects on
tissues -  it is very informative. 

Gayle Callis
HTL(ASCP)HT,MT
Bozeman MT 

 

 
  

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Adam Bazama
Sent: Tuesday, May 19, 2009 3:44 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Cryoprotection Issues with Unfixed Mouse Hearts

Does anyone have any suggestions or protocols for cryoprotecting unfixed
mouse heart tissue? Some of the ones I have been cryosectioning lately have
been damaged, I think because of improper cryoprotection.

 

Thanks!

 

Adam

baza0013 <@t> umn.edu

 

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