[Histonet] RE:The great apoptosis stains debate (TUNEL, Caspases, etc)

[Melissa Gonzalez]

Hi all, 
I have been around this mess a few years back, I gave up on TUNEL long ago for the various reasons mentioned on the list recently. 

Per investigators requests, I have tried several ways to demonstrate cell death, however methods such as PI or Annexin staining are more suitable for whole cells, not tissue sections. I have also not had any luck staining for Cytochrome C. 

We routinely use R&D Systems rabbit anti human/mouse active Caspase3 using a high pH (EDTA) based Ag retrieval in a steamer (enzymes do not work). Works like a charm.

Any other suggestions? I know this topic has been thrown around a lot, but these discussions after all, help us get our jobs done more proficiently.

Melissa


Melissa A. González Edick
R&D, Cell Genesys Inc.
500 Forbes Blvd
South San Francisco, CA 94080
p(650) 266-3168
f (650) 266-3080
 
"It's not enough to believe what you see, you must also understand what you see." -Leonardo Da Vinci
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Message: 6
Date: Tue, 4 Sep 2007 15:18:39 -0400
From: Cheryl Cross <ccross6032 <@t> aol.com>
Subject: Re: [Histonet] TUNEL, Formalin, DNA strand breaks
To: JR R <rosenfeldtek <@t> hotmail.com>
Cc: histonet <@t> lists.utsouthwestern.edu
Message-ID: <4C1E074D-064A-45D1-BD32-13D7E2222B26 <@t> aol.com>
Content-Type: text/plain;	charset=US-ASCII;	delsp=yes;	format=flowed

Hi all -

I have been round and round with this issue myself; I was basically  
told by people who've attempted it that TUNEL on FFPE sections is not  
specific enough due to the formalin issue.

If you are trying to nail apoptosis, what about anti-active  
caspase-3? mind you, that staining can be a bit of a booger too, but  
it should be more sensitive and specific for apoptosis (no references  
to offer, i'm just repeating what i have been told).


Cheryl Cross, DVM, Dipl. ACVP
Researcher
University Corporation for Atmospheric Research
College of Veterinary Medicine
University of Tennessee Department of Pathology
2407 River Drive, Room A201
Knoxville, TN 37996-4542
(423) 967-2724
fax: 865-974-5616
ccross <@t> ucar.edu
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Message: 5
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
(206)-685-7190


From: koellingr <@t> comcast.netTo: rosenfeldtek <@t> hotmail.com; histonet <@t> lists.utsouthwestern.eduSubject: RE: [Histonet] TUNELDate: Fri, 31 Aug 2007 23:17:03 +0000

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