[Histonet] TUNEL, formalin DNA breaks, CLCas3

[koellingr <@t> comcast.net]

I agree,
One of my lab mates in grad school used mouse developing embryos to look at apoptosis in the webbing between the forepaw digits during development.  Those cells don't necrose.  They go through a very specific programmed cell death.  Then he was looking at the macrophages, by IHC, eating the apoptotic debris and whether those macs were resident or homing from other areas of the developing limb bud.  This was before cleaved caspace-3 was available.  His TUNEL was exquisite on formalin fixed mouse embryo's and reproducible for years.  My heart and desire laid in the thymus and how T-lymphocytes undergo development as they survive to express a their T-cell repertoire and develop fully (less than 5%) or apoptose (95%) for a variety of reasons.  His TUNEL procedure didn't work so well on my thymus and my tweaked procedure worked, but not so well on his mouse embryo's.  In a former company, had a post-doc looking at myocardiocytes and a certain type of development and apoptosis they go th
rough.  She had to tweak the procedure a third way for her needs to get really fabulous staining.  As Mesruh said, controls are vital and there are ways that cells can apoptose, not necrose, by alternate pathways than cleaved caspace-3, although it is certainly a very simple and reliable and wonderful IHC stain to do.

Raymond Koelling
PhenoPath Laboratories
Seattle, WA

-------------- Original message -------------- 
From: "mesruh turkekul" <turkekul <@t> gmail.com> 

> Dear All, 
> 
> When I do TUNEL on FFPE mouse or human tumors I always run TUNEL and 
> ClCas3 on FFPE mouse embryos 12.5 days and mouse testis. Since I know 
> exactly where the apoptotic cells are in the embryos and testis I see 
> no difference between TUNEL and ClCas3. In some cases some tumors are 
> positive with TUNEL but not positive with ClCas3 which means that 
> TUNEL maybe also staining necrotic cells which can be identified by 
> their morphology or it may indicate that there are some apoptotic 
> pathways which does not involve activation of ClCas3 but eventfully 
> form DNA breaks detected by TUNEL. 
> If in one paper it is mentioned that formalin creates DNA breaks it 
> does not mean it is true, on the contrary if there is only one paper 
> and nobody reproduced this result after 6 years I will remain 
> sceptical. I always trust papers and I always check their claims by 
> controls. 
> 
> Regards, 
> Mesruh Turkekul 
> MSKCC MCCF 
> www.mskcc.org 
> New York, NY 10021 
> 
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