[Histonet] TUNEL of frozen thick rat brain slices.

[Montina Van Meter]

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-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
Guillermo Palchik
Sent: Wednesday, August 04, 2010 11:21 AM
To: histonet <@t> 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


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