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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Adam Bazama Sent: Tuesday, May 19, 2009 3:44 PM To: histonet@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 baza0...@umn.edu _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet