[Histonet] IHC anti-cleaved caspase 3

Tue Sep 4 20:24:26 CDT 2007

We use Biocare's cleaved caspase-3 (cat# CP229B) at 0.21 ug/ml for 30
minutes.  We use it on formalin fixed paraffin embedded sections after
high pH steam antigen retrieval for 15 minutes with 15 minute cool down.
Trilogy or pH 6.0 citrate buffer also work for antigen retrieval.
Tonsil is a great control.
Good Luck,
Tracy Goodpaster  

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Roger
Moretz
Sent: Tuesday, September 04, 2007 1:33 PM
To: Cheryl Cross; JR R
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] TUNEL, Formalin, DNA strand breaks

Never had the problem with TUNEL on formalin fixed tissue sections.  And
I have also used the anti-cleaved caspase 3 technique.  Both worked
beautifully, but at this point, due to the fact the the IHC anti-cleaved
caspase 3 method is simpler, that's the way I'd go.  I am racking my
retired brain to come up with names of vendors.  So far no joy.  Two
months of retirement (as of today) seem to have put my memory in
neutral.  If I come up with them I'll get back to you.  All the catalogs
and methods are still with my former place of employment....  I have
done the TUNEL stain to verify results, and the pathologist wanted to
compare apples to apples, as it were.  All the new work is being done
with the caspase 3 IHC.

Roger C. Moretz, Ph.D. (Ret.)

--- Cheryl Cross <ccross6032 <@t> aol.com> wrote:

> 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
> 
> 
> 
> 
> 
> On Sep 4, 2007, at 2:46 PM, JR R wrote:
> 
> >
> > 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
> >
> > -------------- 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 >
> > you> > >
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