[Histonet] Caspase3
Melissa Mazan
melissa.mazan <@t> tufts.edu
Thu Sep 6 08:02:38 CDT 2007
Can you elaborate on your high pH EDTA antigen retrieval protocol?
Thanks - Melissa Mazan
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> Today's Topics:
>
> 1. Xylene (themagoos)
> 2. RE:The great apoptosis stains debate (TUNEL, Caspases, etc)
> (Melissa Gonzalez)
> 3. Re: RDO decalcifier (Robert Richmond)
> 4. Re: IHC anti-cleaved caspase 3 (Carl Hobbs)
> 5. avian sperm cell topography (melissah rowe)
> 6. Re: IHC anti-cleaved caspase 3 (Thomas Pier)
> 7. Re: IHC anti-cleaved caspase 3 (Cheryl Cross)
> 8. Fwd: Users of Lab Vision autostainer (Victoria Baker)
> 9. Re: Microwaves VENDOR RESPONSE (Phil McArdle)
> 10. Re: Xylene (Rene J Buesa)
> 11. RE: IHC anti-cleaved caspase 3 (Liz Chlipala)
> 12. Freezing mouse testes for frozen sections
> (Sarah Clatterbuck Soper)
> 13. Olympus BX40 (Mike Pence)
> 14. Look for a used stereoscope (Yu, Jian)
> 15. RE: RDO decal (Tony Henwood)
> 16. PTH antibody reacted with dog tissue... (ChoiUl Soo)
> 17. Floater sources (Michelle McCoy)
> 18. RE: Xylene (Kemlo Rogerson)
> 19. RE: Floater sources (Kemlo Rogerson)
> 20. Fwd: Users of Lab Vision autostainer (Victoria Baker)
> 21. RE: Xylene (Joseph Kapler)
> 22. Unsubscribe (Malam Jacqueline)
> 23. Re: Floater sources (Joe Nocito)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 05 Sep 2007 10:21:51 -0700
> From: "themagoos" <themagoos <@t> rushmore.com>
> Subject: [Histonet] Xylene
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <46dee5af.14b.450a.1030817715 <@t> rushmore.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Can somebody tell me if there are any effects on tissue if
> there is prolonged processing time in xylene?
>
> Jason McGough HT(ASCP)
> Clinical Laboratory of the Black Hills
> Account Representative - Anatomic Pathology
> 2805 5th Street
> Rapid City, SD 57701
> 605-343-2267
> jmcgough <@t> clinlab.com
>
>
>
>
> ------------------------------
>
> Message: 2
> Date: Wed, 5 Sep 2007 11:30:38 -0700
> From: "Melissa Gonzalez" <Melissa.Gonzalez <@t> cellgenesys.com>
> Subject: [Histonet] RE:The great apoptosis stains debate (TUNEL,
> Caspases, etc)
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <2884B897182A1D438C7BA24B9A8F94A20E701D <@t> hqsvr01mail.cgi.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> 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
> ------------------------------
>
> 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
> ------------------------------
>
> 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
>
>
>
> ------------------------------
>
> Message: 3
> Date: Wed, 5 Sep 2007 14:56:14 -0400
> From: "Robert Richmond" <RSRICHMOND <@t> aol.com>
> Subject: [Histonet] Re: RDO decalcifier
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <abea52a60709051156p41ff1c13v25b6efc7f7e2e4bd <@t> mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> I don't see how your hazmats people can tell you what to do to dispose
> of RDO, since nobody has any idea what's in it. It's a murky yellow
> liquid that continuously throws a black sediment that has to be
> filtered out if you're to see your specimen in it. It's sort of the
> ultimate secret formula, and I've always wondered why it's so popular
> - I've had to use it many different places in my travels.
>
> I'd suggest either an ordinary proprietary decalcifier that's a clear
> liquid, probably hydrochloric acid - or else save money and dilute
> your own hydrochloric acid. You can re-use it for a while. When the
> time comes to dispose of it, it can be safely diluted with a lot of
> water and put down the drain.
>
> Remember - it's been stressed on this list many times - that "Decal"
> is a brand name still in use, and that other brands of decalcifying
> solution should not be called Decal.
>
> Bob Richmond
> Samurai Pathologist (never a decalcified Carmelite)
> Knoxville TN
>
>
>
> ------------------------------
>
> Message: 4
> Date: Wed, 5 Sep 2007 20:29:36 +0100
> From: "Carl Hobbs" <carl.hobbs <@t> kcl.ac.uk>
> Subject: Re: [Histonet] IHC anti-cleaved caspase 3
> To: "Histonet" <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <001801c7eff3$18894850$4101a8c0 <@t> carlba65530bda>
> Content-Type: text/plain; format=flowed; charset="iso-8859-1";
> reply-type=original
>
> Hi.
>
> Recent Cleaved caspase 3 posts have not mentioned the antibody details: I
> would be grateful for the source of these Abs.
> Carl
>
>
>
>
> ------------------------------
>
> Message: 5
> Date: Wed, 5 Sep 2007 14:36:50 -0500
> From: melissah rowe <melissah <@t> uchicago.edu>
> Subject: [Histonet] avian sperm cell topography
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <F86819FB-0478-4006-8967-5A8BC257E7B9 <@t> uchicago.edu>
> Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed
>
> Hi,
>
> I am a PhD student working on a behavioral ecology project on
> Australian fairy-wrens. I am currently trying to find out if I can
> determine the dimensions of discrete sections of individual sperm
> cells, i.e. acrosome length, nucleus length, midpiece length and tail
> length using the material I currently have available. I have prepared
> slide smears of sperm that has been previously stained with an eosin-
> nigrosin stain (to determine cell viability) and would like to be
> able to somehow stain different segments of the sperm so that I can
> identify and measure them using an ocular micrometer on a bright
> field or phase contrast microscope set up. It is my understanding
> that I cannot use fluorescent stains given that I have already used a
> colormetric stain (the eosin-nigrosin). If anyone has suggestions for
> ways to identify discrete regions of avian (passerine) sperm cells I
> would be very grateful to hear from you.
>
> Many thanks in advance,
> melissah
>
> -----
> melissah rowe
>
> PhD candidate
> Department of Ecology & Evolution
> University of Chicago
> E. 57th Street, Chicago, IL, 60637
> ph: +1 773-702-3070
> fax: + 1 773-702-9740
>
> email: melissah <@t> uchicago.edu
>
>
>
>
>
> ------------------------------
>
> Message: 6
> Date: Wed, 05 Sep 2007 14:39:04 -0500
> From: "Thomas Pier" <tp2 <@t> medicine.wisc.edu>
> Subject: Re: [Histonet] IHC anti-cleaved caspase 3
> To: <carl.hobbs <@t> kcl.ac.uk>, <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <46DEBF88020000DF0000A736 <@t> gwmail.medicine.wisc.edu>
> Content-Type: text/plain; charset=US-ASCII
>
> Cell Signalling Technology has a good rabbit monoclonal for Cleaved Caspase-3.
>
> Tom Pier
>
>
>>>> "Carl Hobbs" <carl.hobbs <@t> kcl.ac.uk> 09/05/07 2:29 PM >>>
>>>>
> Hi.
>
> Recent Cleaved caspase 3 posts have not mentioned the antibody details: I
> would be grateful for the source of these Abs.
> Carl
>
>
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>
>
>
>
> ------------------------------
>
> Message: 7
> Date: Wed, 5 Sep 2007 15:54:35 -0400
> From: Cheryl Cross <ccross6032 <@t> aol.com>
> Subject: Re: [Histonet] IHC anti-cleaved caspase 3
> To: "Thomas Pier" <tp2 <@t> medicine.wisc.edu>
> Cc: carl.hobbs <@t> kcl.ac.uk, histonet <@t> lists.utsouthwestern.edu
> Message-ID: <E3C972DF-6E5F-4FD1-8096-30C930BB47E0 <@t> aol.com>
> Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed
>
> I will second the Cell Signaling antibody - we have tried antibodies
> from Promega (which gave tons of background staining)...then via this
> site i was pointed to Cell Signaling's monoclonal which we tried; the
> polyclonal actually has been working very well with minimal
> background (we were getting lots of respiratory epithelium and
> endothelium lighting up in control animals). I have been using it in
> mice with this protocol:
>
> EDTA with PASCAL
> primary antibody 60 minutes, dilution 1:125
> Rabbit envision
> DAB plus
>
> Hope this helps!
>
> 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
>
>
>
>
>
>
>
>
> ------------------------------
>
> Message: 8
> Date: Wed, 5 Sep 2007 15:59:45 -0400
> From: "Victoria Baker" <bakevictoria <@t> gmail.com>
> Subject: [Histonet] Fwd: Users of Lab Vision autostainer
> To: "Histo Net list server" <HistoNet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <4f016b690709051259rcdffccet70d7f97ae0457707 <@t> mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> One more try, first one didn't go through it seems! Thanks
>
> ---------- Forwarded message ----------
> From: Victoria Baker <bakevictoria <@t> gmail.com>
> Date: Sep 5, 2007 9:02 AM
> Subject: Users of Lab Vision autostainer
> To: Histo Net list server <HistoNet <@t> lists.utsouthwestern.edu>
>
>
> Hi
>
> I'm a new user of the Lab Vision autostainer and I'm looking to see if
> I can find users in Histo-land that have experience with it. The
> facility only works with human tissue and all of the antibodies are
> for dx purposes.
>
> My key questions are as follows:
> How many antibodies is your lab running?
> How many users do you allow?
> How many people do you allow programming rights and at what level of
> supervision are they?
>
> How many of these antibodies are from Lab Vision?
> a) are they concentrates or pre-dilutes?
> b) for HIER are you using their PT modules/procedures or
> your own equipment (microwave, steamer, pressure cooker etc) and in
> house designed protocols for retrieval?
> c) for digestion do you only use their Pro-K or have you
> designed your own in-house methods using other reagents for digestion?
> d) do you put your controls on the same slide as the patient?
> e) are your controls in-house or commercial?
> f) do you have more than one stainer hooked up to one computer system?
>
> What Version of software do you currently have on your system?
>
> Any feed back would be very helpful.
>
> Thanks in advance.
>
> Vikki Baker
> Interim Histology Manager
> Mission Hospital System
> Asheville, NC
>
>
>
> ------------------------------
>
> Message: 9
> Date: Wed, 05 Sep 2007 16:00:47 -0400
> From: Phil McArdle <PMcArdle <@t> ebsciences.com>
> Subject: Re: [Histonet] Microwaves VENDOR RESPONSE
> To: Joe Nocito <jnocito <@t> satx.rr.com>
> Cc: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <46DF0AEF.7030203 <@t> ebsciences.com>
> Content-Type: text/plain; charset=UTF-8; format=flowed
>
> Hi Joe:
>
> Obviously, a microwave vendor hates to hear microwave horror stories, so
> again, no argument - even though I'm not privy to details of the fried
> biopsies in question or what type/vintage of microwave, anyone who's
> experienced a malfunction involving patient samples doesn't want a
> repeat performance. And pathology is, must be, risk averse.
>
> That said, again, any mechanical or electronic equipment can fail, or
> user error can contribute; just look at the "hang-up" problems with
> older tissue processors that are now ancient history. It's up to
> manufacturers to minimize the possibilities of failure, since patient
> care is at stake. Improvement is therefore a continual, ongoing process.
> For example, while for years EBS microwave processors incorporated
> safety shutdown modes in the event of vent failure, probe failure (open
> and closed) and many other component-related issues, about two years ago
> we determined that the microwave did not have a comprehensive set of
> safeguards to deal with user errors, for example, temperature overshoots
> caused by too small a container for a given power setting, or failure to
> place the temperature probe in solution. So we developed multiple safety
> mechanisms to head off user errors of this sort.
>
> PMM
>
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