Usually the cryoprotection is carried out after the specimens are fixed and before they are frozen. If you have a sample you can spare you might try making up some 20% Sucrose placing the frozen sample in it. putting it in the refrigerator and letting it stay in the 20% sucrose until it drops to the bottom of the container. You might have to change the 20% Sucrose a couple of times as you know sucrose can grow bacteria easily. When the specimen has dropped to the bottom of the container, remove it, rinse in a couple of changes of distilled water and quick freeze the sample. Try cutting and staining the sample and see if the morphology is good if it is then you can do all of your samples to correct the problem. There should be no thawing between removal of the sample from the storage to the cryostat. Hope this helps Frances Swain
________________________________________ From: histonet-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Dr. med. Frauke Neff [ne...@staff.uni-marburg.de] Sent: Wednesday, March 18, 2009 3:47 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] cryoprotection possible? Dear Histonetters, I'm supposed to do a noradrenaline ihc on rat brains that have been snap frozen and afterwards glutaraldehyd fixed. While the morphology is okay in the "test"-tissue we used to establish the protocol, it was very poor in the tissue of the trial animals. It appeared mooth eaten and disrupted. I assume the brains were thawed and refrosted due to moving the samples between several cities. Does anyone know a method /a tip how I can save some proper morphology? I checked the archive and found sucrose as cryoprotectant, but this is supposed to do it while the tissue is frozen or is it possible to use it while the tissue thaws? Thank you all in advance for your patience and help, Frauke -- Dr. med. Frauke Neff AG Neurologische Therapieforschung Neurologie Philipps-Universität Marburg Rudolph-Bultmann Str. 8 35039 Marburg 06421/5866304 ---------------------------------------------------------------- This message was sent using IMP, the Internet Messaging Program. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Confidentiality Notice: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet