[Histonet] TUNEL

[Bernice Frederick]

You could probably run a Survivin antibody for comparasion.
Bernice

Bernice Frederick HTL (ASCP)
Northwestern University
Pathology Core Facility
710 N Fairbanks Court
Olson 8-421
Chicago,IL 60611
312-503-3723


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
koellingr <@t> comcast.net
Sent: Friday, August 31, 2007 6:17 PM
To: JR R; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] TUNEL

Jerry,
Could you expand on or give references to formalin causing DNA strand
breaks?  Double strand breaks, single strand, blunt end, overhanging?  My
pile of papers and having done TUNEL for years says that formalin fixation
is a very good technique for TUNEL and many peer-reviewed articles in which
TUNEL is used as a technique, use formalin fixation and how can that be if
formalin is causing strand breaks?  In fact one paper I'm looking at says
that extended (5-7 weeks in formalin) fixation causes loss of TUNEL signal.
If formalin is causing breaks, you would assume that TUNEL pos signals would
increase with extended formalin fixation.  Even the use of the monoclonal
antibody F7-26, for single stranded DNA, touts formalin fixation for their
claims of discriminating apoptosis from necrosis.

The question asks about extended alcohol fixation but your answer is
possibly a lot of false positives because formalin causes DNA breaks.  Does
this imply that alcohol won't?  Have done a lot of TUNEL on alcohol fixed
samples.  True I couldn't pretreat them and handle them they way I would
handle FFPE tissue.  Also true that we could argue specificity and ability
or not to discriminate apoptosis from necrosis for quite a while.  But if
formalin itself is causing the breaks in DNA, I and a lot of people are in
big trouble with our science projects and experiments.  Also I can't
envision why 2 cells are showing TUNEL positivity while 2 of the same type
of cells right next to them (and getting the same formalin fix), are
absolutely clean and there is no background?

Thanks for any information you can provide.

Ray Koelling
PhenoPath Laboratories
Seattle, WA

-------------- Original message -------------- 
From: JR R <rosenfeldtek <@t> hotmail.com> 

> 
> I expect you will get a very high background--lots of false positives.
That's a 
> problem with TUNEL and formalin fixed tissue anyway--formalin causes DNA
strand 
> breaks. 
> 
> 
> 
> Jerry L. Ricks 
> Research Scientist 
> U.W. Medicine at South Lake Union 
> 815 Mercer Street 
> Seattle, WA 98109 
> (206)-685-7190> Date: Fri, 31 Aug 2007 10:42:32 +0200> From: 
> marijke.oste <@t> ua.ac.be> To: histonet <@t> lists.utsouthwestern.edu> Subject: 
> [Histonet] TUNEL> > For staining the apoptotic cells I'm already using a
kit of 
> Roche> diagnostics. I think the greatest problem is that my tissues has
been 
> saved> for several years in ethanol 70%. Does anyone has experience in
staining> 
> tissues for apoptotic cells who are kept in ethanol for several years?>
Thank 
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