[Histonet] TUNEL assay validity issues

[koellingr <@t> comcast.net]

Seema,
I agree that you cannot necessarily distinguish an apoptotic from necrotic event from another DNA damaging event and be 100% confident in the TUNEL readout.  DNA breaks are DNA breaks and they are there for any number of molecular reasons.  TUNEL isn't and can't be a gold standard. On the other hand TUNEL, taken with a bit of common sense and with other observations can I think be a useful tool.  If you are looking at mouse spinal cord and seeing what appear to be some TUNEL positive events, what are the possibilities?  Could it truly be an apoptotic event?  Sure, depending on your mouse model and study.  Might it be necrotic?  Sure but do you see any other evidence of necrosis around there?  Might it be procedurally induced from the surface of a section?  Sure but then why are 10 nuclei staining and the 100's around them, which are also on a cut surface, not staining?  Also remember that not all apoptosis follows a single canonical molecular pathway.   TUNEL or activated caspace-3
 positivity is simply not ultimately pathognomonic of an apoptotic event.  It would be like relying 100% on a single antibody to make a 100% definitive diagnosis on a tumor when in fact a panel of several antibodies is necessary to create a sufficient weight of evidence to raise your confidence to the highest level.

Ray
Almost, but not quite yet, employed in Seattle, WA

-------------- Original message -------------- 
From: "Seema Shroff" <shroffsm <@t> vcu.edu> 

> Hi everyone, 
> 
> 
> 
> I am a grad student doing fluorescently labeled TUNEL assay on mouse spinal 
> cord frozen sections. I used 10 day mouse liver as my positive control and 
> I'm pretty sure that my technique works. But I have a question - The TUNEL 
> enzyme does not distinguish between apoptotic DNA breaks and DNA physically 
> broken (especially on the surfaces of the section) due to the cryostat knife 
> slicing through the tissue block. 
> 
> 
> 
> The Roche technical support confirmed this. TUNEL is such a widely used 
> technique and well-accepted. Can anyone shed light on this doubt? Is it 
> really of concern and if yes, what can I do to validate this method? 
> 
> 
> 
> Thank you, 
> 
> 
> 
> Seema. 
> 
> 
> 
> 
> 
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