[Histonet] TUNEL

[koellingr <@t> comcast.net]

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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