Hi Roger & Cheryl, Yeah I think I will try to go with the activated Caspase 3 stain. I just checked the Roche site and found that for TUNEL staining some tissues that give high false positives, one ought to use a Citrate buffer (pH 6.0) antigen retrieval step instead of a proteinase k digest. Apparently sacrificing a chicken under the full moon works sometimes, too.
Jerry L. Ricks Research Scientist U.W. Medicine at South Lake Union 815 Mercer Street Seattle, WA 98109 (206)-685-7190 ----------------------------------------> Date: Tue, 4 Sep 2007 13:32:44 -0700> From: firstname.lastname@example.org> To: email@example.com; firstname.lastname@example.org> Subject: Re: [Histonet] TUNEL, Formalin, DNA strand breaks> CC: email@example.com>> 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 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>> firstname.lastname@example.org>>>>>>>>>>>> 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: email@example.comTo:>> firstname.lastname@example.org;>>> email@example.comSubject: 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>>>>> 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:>>>> firstname.lastname@example.org> To:>> email@example.com>>>> 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>>>>> _______________________________________________>>> Histonet>>> mailing list>> Histonet@lists.utsouthwestern.edu>>>> http://>>> lists.utsouthwestern.edu/mailman/listinfo/histonet>>>>>>>>> _________________________________________________________________>>>>>> Explore the seven wonders of the world>>> http://search.msn.com/>>>>>> results.aspx?q=7+wonders+world&mkt=en-US&form=QBRE________>>>>>> _______________________________________> Histonet>> mailing list>>>> Histonet@lists.utsouthwestern.edu>>> http://lists.utsouthwestern.edu/>>> mailman/listinfo/histonet>>>>>> _________________________________________________________________>>> Discover the new Windows Vista>>>>>> http://search.msn.com/results.aspx?q=3Dwindows+vista&mkt=en->>>>>>>> US&form=QBRE_______________________________________________>>> === message truncated ===>>>>> ____________________________________________________________________________________> Boardwalk for $500? In 2007? Ha! Play Monopoly Here and Now (it's updated for today's economy) at Yahoo! Games.> http://get.games.yahoo.com/proddesc?gamekey=monopolyherenow>> _______________________________________________> Histonet mailing list> Histonet@lists.utsouthwestern.edu> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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