[Histonet] Re: floating section incubation times

[David A. Wright]

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.VanMeter <@t> 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 <@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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