David, The reason that I am able incubate overnight is that I antigen retrieve my free-floating sections in the Biocare Decloaker (and no, I am not receiving any subsidies from them). This has enabled me to reduce incubation times as well as revealing receptor staining that has been difficult to observe in the past due to the lack of sensitive techniques. I am serum-free and use polymers for detection. This has drastically cut down on my background issues.
Regards, Tina -----Original Message----- From: David A. Wright [mailto:d...@uchicago.edu] Sent: Thursday, August 05, 2010 3:46 PM To: histonet@lists.utsouthwestern.edu Cc: Montina Van Meter; Guillermo Palchik Subject: Re: floating section incubation times Hi Histonet, Tina & Gil I work on the same material as Tina and just wanted to echo her "overnight" comments with the addition that I routinely incubate 40um floating sections over the whole weekend at room temp (on a very slow shaker, 20 rpm, and with azide to stop bug growth. It all washes out before the peroxidase reaction.) I seem to remember from Fick's Law that the time taken to diffuse to the same endpoint concentration goes up with the square of the thickness, so if you double the thickness you would need 4x the incubation time - or else more concentrated antibody. The same distinction applies to "floating" vs on-slide incubations. {It wasn't clear to me if Gil's sections were floating or not). Since reagents can penetrate from both sides of a floating section, a section stained "on-slide" is effectively twice as thick and needs 4x the time. Note that the same principle applies to leaching out unbound reagents - you should lengthen your wash steps too, by the same factor, to prevent high background. happy staining -David David A. Wright, PhD University of Chicago Section of Neurosurgery ---- Original message ---- Date: Thu, 05 Aug 2010 12:07:03 -0500 (CDT) Subject: Histonet Digest, Vol 81, Issue 5 Message: 1 Date: Wed, 4 Aug 2010 12:18:03 -0500 From: "Montina Van Meter" <montina.vanme...@pbrc.edu> Subject: RE: [Histonet] TUNEL of frozen thick rat brain slices. 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----- 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