[Histonet] TUNEL, Formalin, DNA strand breaks

From:JR R

Hi Ray,
Sheesh-- I read (and then lost) the paper maybe 5-7 years ago, back when I was tearing my hair out over high background, or false positives for TUNEL stain in formalin fixed, paraffin embedded arterial sections.  I can troubleshoot any immunostain, but the TUNEL and ISEL assays bedeviled me.  
The paper was titled something to the effect of "formaldehyde causes single and double stranded DNA breaks," and was pretty emphatic.  I'll look for the article.
I think the assay could work in whole cells for flow, or maybe in frozen tissue.  My hunch at this point is that this is one of those rare cases where lots and lots of peer reviewed articles are just plain wrong.  
Hey, if someone has a good protocol, I'd be willing to try it out, and I would be delighted if I turn out to be mistaken.  For now, I think TUNEL is the assay of the beast.
Oh, here is a thought--say you are looking at a 5 micron section through a nucleus.  The microtome blade pretty much had to create a lot of double stranded DNA breaks, no?

Jerry L. Ricks
Research Scientist
U.W. Medicine at South Lake Union
815 Mercer Street
Seattle, WA 98109

From: koellingr@comcast.netTo: rosenfeldtek@hotmail.com; histonet@lists.utsouthwestern.eduSubject: RE: [Histonet] TUNELDate: Fri, 31 Aug 2007 23:17:03 +0000
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  > > 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@ua.ac.be> To: histonet@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 > you> > > _______________________________________________> Histonet mailing list> > Histonet@lists.utsouthwestern.edu> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > _________________________________________________________________ > Explore the seven wonders of the world > http://search.msn.com/results.aspx?q=7+wonders+world&mkt=en-US&form=QBRE________ > _______________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
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