Forgot to mention that we keep the cryostat around -16 to -18 C Brett
-----Original Message----- From: Connolly, Brett M Sent: Thursday, November 17, 2011 2:58 PM To: 'McLaughlin, Terry '; histonet@lists.utsouthwestern.edu Subject: RE: to cut or not to cut mouse brains Terry, Here what works for us - Slides are kept prechilled in a -20C freezer and then kept cold in the cryostat. Very gently press the cold slide onto the cut section so it makes contact and the section sticks to the slide. Flip slide over and use your finger to warm the back of the slide under the section. Starting with cold slides is a must for us. Brett Brett M. Connolly, Ph.D. Imaging Research Fellow Merck & Co., Inc. PO Box 4, WP-44K West Point, PA 19486 brett_conno...@merck.com T- 215-652-2501 F- 215-993-6803 -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of McLaughlin, Terry Sent: Thursday, November 17, 2011 2:25 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] to cut or not to cut mouse brains Hello All, I am having trouble cutting frozen mouse brains and was wondering if someone can offer some help. The mouse was perfused in 4% PFA , the brain removed and kept in PFA for 22 hrs. We placed it in 30% sucrose for 20 hrs, until the brain sank in this solution. It was frozen back by using a culture plate floating in liquid nitrogen with OCT . The problem I am having is many folds, and air bubbles under the tissue. What I have done so far: Cut at temp of -25, then tried - 22 and -17. Cut at 10um, then tried 16-25um. Tried making the knife colder by adding dry ice to it for a few seconds. Added dry ice to sample for a couple seconds. The sample cuts fine, it appears to happen when picking up on a slide. I tried picking up by starting at one side or end and letting tissue spread onto slide. Did not work. Also tried gently laying slide on top of sample and removing gently-still folds and air bubbles. Any help would be greatly appreciated as we have 6 more to do. Signed, Help! Terry Kokas _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet