Gil, I routinely incubate free-floating 40um rat brain sections overnight or longer @ 4 degrees centigrade according to the particular antibody that I am working with. Our sections are perfused in 4% paraformaldehyde and cryoprotected with 30% sucrose prior to sectioning. How long are you fixing the tissue prior to staining?
Tina -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Guillermo Palchik Sent: Wednesday, August 04, 2010 11:21 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] TUNEL of frozen thick rat brain slices. Dear Histologists, Has anyone done TUNEL on 40 um flash-frozen rat brain sections? I have tried tweaking the protocol (we use the Millipore Apoptag kit - S7101) from our ongoing 20 um slices by increasing the incubation times from 1 hour to 2 and 4 hours but we still get poor staining. Also, could I do overnight incubations? the tissue is flash frozen, but it does get fixed at the beginning of the protocol with PFA (4%) and subsequently with acetic acid-EtOH. Thanks, Gil -- Guillermo Palchik Ph.D. Candidate - Interdisciplinary Program in Neuroscience Georgetown University Medical Center Research Building Room W 217 3970 Reservoir Rd. NW, Washington, DC 20007 Lab: 202-687-7825 _______________________________________________ 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