[Histonet] RE: Histonet Digest, Vol 20, Issue 5
Joe, I could not agree with you more, in these days of declining revenue and increasing costs, it seems almost criminal to discard a reagent in histology because of an arbitrary date. Having once worked for a antibody manufacturer, we determined expiration dates by subjecting the Ab to extreme high and low temperatures to accelerate the rate at which the reagent would stop working. In routine circumstances in a lab, this would not be the case. In any event, as long as the reagent passes QC each time it is used should be the determining point. Since CAP does not pay for my reagents, I feel as you do that the question should be revisitedMike Ricepatholgy supervisorholy cross hospitalft lauderdale-----Original Message-----From: histonet-bounces@lists.utsouthwestern.edu[mailto:histonet-bounces@lists.utsouthwestern.edu]On Behalf Ofhistonet-request@lists.utsouthwestern.eduSent: Tuesday, July 05, 2005 1:06 PMTo: histonet@lists.utsouthwestern.eduSubject: Histonet Digest, Vol 20, Issue 5Send Histonet mailing list submissions to histonet@lists.utsouthwestern.eduTo subscribe or unsubscribe via the World Wide Web, visit http://lists.utsouthwestern.edu/mailman/listinfo/histonetor, via email, send a message with subject or body 'help' to histonet-request@lists.utsouthwestern.eduYou can reach the person managing the list at histonet-owner@lists.utsouthwestern.eduWhen replying, please edit your Subject line so it is more specificthan "Re: Contents of Histonet digest..."Today's Topics: 1. PCR and ISH in fixed tissues (Caroline Bass) 2. Re: PCR and ISH in fixed tissues (Agripina Suarez) 3. Re: formic acid decal (clifford berger) 4. RE: PCR and ISH in fixed tissues (Tony Henwood) 5. ER/PR staining of normal breast tissue (Patrick Paulusse) 6. non-muscle myosin IIA (Edwards, R.E.) 7. Rotating shifts... (Tom McNemar) 8. antibody search for BMP (louise renton) 9. revisiting expired immmuno reagents (Joe Nocito) 10. RE: Formic Acid decalcification (Pixley, Sarah (pixleysk)) 11. Impress (Van Eyck, Deb) 12. liver cryosections (Till, Renee) 13. RE: liver cryosections (Monfils, Paul)----------------------------------------------------------------------Message: 1Date: Mon, 04 Jul 2005 13:54:08 -0400From: Caroline Bass Subject: [Histonet] PCR and ISH in fixed tissuesTo: 'histonet@lists.utsouthwestern.edu' Message-ID: <9E94278E-ECB4-11D9-8BC8-0003930771EE@bidmc.harvard.edu>Content-Type: text/plain; charset=US-ASCII; format=flowedHey guys,My boss is submitting a grant and he does not believe that you can do PCR (RT) or ISH with fixed tissues. I thought you could but I don't know any details. Could someone elaborate? Is there a particular way the tissue should be fixed? In other words do I have to use FFPE sections or can I use 4% paraformaldehyde perfusion?Any advice would be helpful.Thanks,Caroline Bass------------------------------Message: 2Date: Mon, 04 Jul 2005 11:09:57 -0700From: "Agripina Suarez" Subject: Re: [Histonet] PCR and ISH in fixed tissuesTo: ,Message-ID: Content-Type: text/plain; charset=US-ASCIIHi Caroline.Most of the ISH I do are on paraformaldehyde/formalin-fixedparaffin-embedded sections and they work very well. The tissues arefixed either in 4% paraformaldehyde or in commercially available 10%neutral buffered formalin for 12 to 24 hours, depends on the size of thetissue.Hope this helps.AgripinaAgripina C. SuarezMcDonald Research LaboratoriesiCAPTURE Centre, St Paul's Hospital1081 Burrard St, VancouverB.C., Canada V6Z 1Y6Tel no. 604 682 2344 local 62562Fax no. 604 806 8351>>> Caroline Bass 7/4/2005 10:54:08 AM >>>Hey guys,My boss is submitting a grant and he does not believe that you can do PCR (RT) or ISH with fixed tissues. I thought you could but I don't know any details. Could someone elaborate? Is there a particular waythe tissue should be fixed? In other words do I have to use FFPE sections or can I use 4% paraformaldehyde perfusion?Any advice would be helpful.Thanks,Caroline Bass_______________________________________________Histonet mailing listHistonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet------------------------------Message: 3Date: Mon, 04 Jul 2005 16:10:06 -0400From: clifford berger Subject: Re: [Histonet] formic acid decalTo: patsy ruegg , lpwenk@sbcglobal.net, 'louise renton' , Histonet@lists.utsouthwestern.eduMessage-ID: <000e01c580d4$5f1e71e0$0200a8c0@dellovo0ll7kuk>Content-Type: text/plain; charset=iso-8859-1For more information on this product, technical papers and much more information, you can visit our website, www.decal-bone.com. You can also call us at 1-800-428-5856 for free samples. Best regards and happy 4rthCliff BergerDecal Chemical CorpThe Real Decal1-800-428-5856----- Original Message ----- From: "patsy ruegg" To: ; "'louise renton'" ; Sent: Monday, July 04, 2005 11:24 AMSubject: RE: [Histonet] formic acid decal> For I IHC I use ImmunoCal for Decal Chemicals, it is a 5% formic acid with> buffers.> Patsy> > -----Original Message-----> From: histonet-bounces@lists.utsouthwestern.edu> [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Lee & Peggy> Wenk> Sent: Friday, July 01, 2005 5:13 PM> To: 'louise renton'; Histonet@lists.utsouthwestern.edu> Subject: RE: [Histonet] formic acid decal> > A milder formic acid decalcification solution is FASC = formic acid-sodium> citrate. It's a buffered formic acid. Sections of vertebral bone take 5-7> days to decalcify. I don't know how long a bone biopsy takes. When my> students have to submit a bone section for their certification exam, this is> what we use. It gives great nuclear detail, fantastic eosinophil granules.> So I expect IHC would be equally great. We use the procedure out of the> Bancroft book.> > 65 mL of 20% aqueous trisodium citrate (13 g in 65 mL water)> 35 mL of 90% formic acid (that's full strength. Don't dilute)> pH is about 2.3> Change solution every 3 days.> > We did have a rare bone tumor, and none of the IHC worked, until we> decalcified it in EDTA. Then it worked great. (Sorry, don't remember what> type of tumor or what type of antibody.) But you might want to try the EDTA,> if you have a lot of time. Vertebral bone takes 2-3 weeks. The little bone> fragments we had from the bone tumor took 3 days. (would have been a 2-3> hours in our usual 5% hydrochloric acid)> > 25 g EDTA disodium salt (ethylenediaminetetracetic acid) (note: MUST be> disodium salt)> 175 mL d. water> Solution is cloudy. Add 2.5-3.0 g of sodium hydroxide until solution becomes> clear. pH should be about 7 at that point.> Change solution about twice a week.> > Peggy A. Wenk, HTL(ASCP)SLS> William Beaumont Hospital> Royal Oak, MI 48073> > > Lee & Peggy Wenk> -----Original Message-----> From: histonet-bounces@lists.utsouthwestern.edu> [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of louise> renton> Sent: Friday, July 01, 2005 8:32 AM> To: Histonet@lists.utsouthwestern.edu> Subject: [Histonet] formic acid decal> > Hi all,> > on pain of being repetitive/lazy/slovenly/slothful/unprofessional,> would someone PLEASE share their recipe for formic acid decalcifying> solution suitable for samples on which IHc will be performed. Many many> thanks> > --> Louise Renton (grovelling)> Bone Research Unit> University of the Witwatersrand> Johannesburg> South Africa> "....I know who I am. No-one else knows who I am. If I was a giraffe, and> someone said I was a snake, I'd think no, actually I'm a giraffe"> Richard Gere> > _______________________________________________> Histonet mailing list> Histonet@lists.utsouthwestern.edu> http://lists.utsouthwestern.edu/mailman/listinfo/histonet> > > _______________________________________________> Histonet mailing list> Histonet@lists.utsouthwestern.edu> http://lists.utsouthwestern.edu/mailman/listinfo/histonet> > > _______________________________________________> Histonet mailing list> Histonet@lists.utsouthwestern.edu> http://lists.utsouthwestern.edu/mailman/listinfo/histonet------------------------------Message: 4Date: Tue, 5 Jul 2005 09:10:29 +1000From: Tony Henwood Subject: RE: [Histonet] PCR and ISH in fixed tissuesTo: "'Caroline Bass'" , "'histonet@lists.utsouthwestern.edu'" Message-ID: <1CF2E2E5BB36D5119E7A0008C791F3741269E2C0@simba.kids>Content-Type: text/plainCaroline,We have successfully done ISH for EBERs, Adenovirus RNA, CMV RNA,Chromogranin mRNA, Estrogen Receptor mRNA, and nCAM RNA to name a few onboth Frozen sections and Formalin Fixed Paraffin Embedded Tissue (FFPE).We have also successfully extracted RNA and DNA from FFPE tissues for PCR(Ewing's and Rhabdo translocation PCR studies)and do this routinely.RegardsTony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)Laboratory Manager & Senior ScientistThe Children's Hospital at Westmead,Locked Bag 4001, Westmead, 2145, AUSTRALIA.Tel: 612 9845 3306Fax: 612 9845 3318-----Original Message-----From: Caroline Bass [mailto:cbass@bidmc.harvard.edu] Sent: Tuesday, 5 July 2005 3:54 AMTo: 'histonet@lists.utsouthwestern.edu'Subject: [Histonet] PCR and ISH in fixed tissuesHey guys,My boss is submitting a grant and he does not believe that you can do PCR (RT) or ISH with fixed tissues. I thought you could but I don't know any details. Could someone elaborate? Is there a particular way the tissue should be fixed? In other words do I have to use FFPE sections or can I use 4% paraformaldehyde perfusion?Any advice would be helpful.Thanks,Caroline Bass_______________________________________________Histonet mailing listHistonet@lists.utsouthwestern.eduhttp://lists.utsouthwestern.edu/mailman/listinfo/histonet**********************************************************************This email and any files transmitted with it are confidential andintended solely for the use of the individual or entity to whom theyare addressed. If you are not the intended recipient, please delete it and notify the sender.Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of TheChildren's Hospital at WestmeadThis footnote also confirms that this email message has been virus scanned and although no computer viruses were detected,the Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses.**********************************************************************------------------------------Message: 5Date: Sat, 4 Jun 2005 20:06:38 -0400From: "Patrick Paulusse" Subject: [Histonet] ER/PR staining of normal breast tissueTo: "Histonet" Message-ID: <000701c56962$85217100$57b05bd1@wolf>Content-Type: text/plain; charset="iso-8859-1"Good Evening All,Should normal breast duct tissue always stain positively with estrogen andprogesterone antibodies. If not, is this a natural occurrence or is theresomething physiologically occuring to prevent the staining. I have hadseveral occasions when the normal breast duct tissue will not stain (thecorresponding tumour tissue in the same block was also negative). Anexternal control on the same slide stains wonderfully. Fixation is neverless than 24 hours and seldom over 72 hours in 10% NBF. I have severalcontrol blocks and use the one that mimics the fixation and processing ofthe test specimen. Any comments and references are always welcome.Thanking you in advance,Patrick PaulusseAnatomical PathologyPembroke Regional Hospital------------------------------Message: 6Date: Tue, 5 Jul 2005 12:32:03 +0100From: "Edwards, R.E." Subject: [Histonet] non-muscle myosin IIATo: Message-ID: Content-Type: text/plain; charset="iso-8859-1"Looking for an antibody to the above for immunohistochemistry... Thanks Richard Edwards MRC TOX UNIT.....LEICESTER.....U.K........------------------------------Message: 7Date: Tue, 5 Jul 2005 09:25:27 -0400 From: Tom McNemar Subject: [Histonet] Rotating shifts...To: Histonet Message-ID: <6CD94D97ED7D924BA5C2B588FA9528213967D9@nt_exchange.lmhealth.org>Content-Type: text/plain; charset="iso-8859-1"I was wondering if most histo labs use rotating shifts or if people mostlywork the same hours. Do you have people that just work one shift andprimarily do one job (gross, cut, stain, etc.)?At full staff we have 4 techs and 3 of them rotate between 2 weeks of 6-2:30and 1 week of 7:30-4 (surgicals). I wondered if switching the hours to astraight 7:30 - 4 position might help attract someone. I was also thinkingthat we would have a more consistency/continuity if everyone had a certainjob/area that they were primarily responsible for. All thoughts appreciated.Tom Mc Nemar HT(ASCP)Histology SupervisorLicking Memorial HospitalNewark, Ohio 43055------------------------------Message: 8Date: Tue, 5 Jul 2005 15:52:42 +0200From: louise renton Subject: [Histonet] antibody search for BMPTo: Histonet@lists.utsouthwestern.eduMessage-ID: Content-Type: text/plain; charset=ISO-8859-1Dear all,I am looking for antibodies to BMP (bone morphogenic protein) 3, 6 and7 (AKA Op1) for use on FFPE embedded tissue. I would be grateful forany input, as an internet search has come up with very littleinformation. Thank you-- Louise RentonBone Research UnitUniversity of the WitwatersrandJohannesburgSouth Africa"....I know who I am. No-one else knows who I am. If I was a giraffe,and someone said I was a snake, I'd think no, actually I'm a giraffe"Richard Gere------------------------------Message: 9Date: Tue, 5 Jul 2005 09:01:51 -0500From: "Joe Nocito" Subject: [Histonet] revisiting expired immmuno reagentsTo: "Histonet" Message-ID: Content-Type: text/plain; charset="iso-8859-1"Hello histoland, okay, I've been shooting my mouth off a lot lately and I want y'all to knowthat I contacted the CAP about revising the question about using expiredimmuno reagents. The question in question (like that huh?) in the latest CAP checklist isANP.22432. Whether they contact me or not is another question (so manyquestions). My rationale for changing the question are these1. Immuno reagents are too expensive to throw away.2. Each time a run is performed, the reagents are revalidated3. A laboratory must have a written procedure in place detailing therevalidating process.4. A quality control form must be used and signed by the medical directorassuring that the reagents work.I am open to suggestions and other ideas that I may have over looked. Withyour support, maybe we can, at least, have this question revised.Of course, I expect some fallout because of this, but hey, it wouldn't be meif I didn'tEnjoy!!!Joe Nocito, BS, HT(ASCP) QIHCHistology ManagerPathology Reference LabSan Antonio, TX------------------------------Message: 10Date: Tue, 5 Jul 2005 10:25:23 -0400 From: "Pixley, Sarah (pixleysk)" Subject: [Histonet] RE: Formic Acid decalcificationTo: "'histonet@lists.utsouthwestern.edu'" Message-ID: Content-Type: text/plainDear Louise Renton:Here is a method from a research lab. We use 10% formic acid, but add an ionexchange resin that soaks up the calcium. This eliminates the need to changesolutions, but brings its own problems since the resin must be stirred. Thisis faster than EDTA, but I don't know if it is faster than just a 10% formicacid solution alone. The mixture is good for many decalcifications. GreatICC staining. Formic Acid Decalcification: Purchase (i.e., Fisher Scientific): Rexyn 101 H ion exchange resin formic acid (full strength, ~90%).Make 6-10 liters of 10% formic acid (in double distilled water)Add 500 g of resin(So ~50-83 g resin per liter of 10% formic acid)Need stir bar to keep resin suspended, so put samples in cloth bags (i.e.,homemade cotton bags) and suspend from side. Alternatively, use labdessicator. Samples sit above dessicator plate while stir bar goesunderneath.Sincerely,Sarah Pixley, Ph.D.Dept. of Cell Biology, Neurobiology and AnatomyUniversity of Cincinnati College of MedicineCincinnati, OH 45267-0521------------------------------Message: 11Date: Tue, 5 Jul 2005 09:28:07 -0500 From: "Van Eyck, Deb" Subject: [Histonet] ImpressTo: "'histonet@lists.utsouthwestern.edu'" Message-ID: Content-Type: text/plain; charset="iso-8859-1"Dear-histonetters-----I have also wondered about the Impress Kit----Amos andto otheres who tried it---was it pretty interchangeable- time wise with theEnvision , or Envision + also what about antibody dilutions? same strength?Another immuno question for you all? is anyone doing Toxoplasmosisstaining? What antibody are you using? What protocol? Thanks much-Deb VanEyck> -----Original Message-----> From: histonet-bounces@lists.utsouthwestern.edu> [SMTP:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of> histonet-request@lists.utsouthwestern.edu> Sent: Sunday, July 03, 2005 12:06 PM> To: histonet@lists.utsouthwestern.edu> Subject: Histonet Digest, Vol 20, Issue 3> > Send Histonet mailing list submissions to> histonet@lists.utsouthwestern.edu> > To subscribe or unsubscribe via the World Wide Web, visit> http://lists.utsouthwestern.edu/mailman/listinfo/histonet> or, via email, send a message with subject or body 'help' to> histonet-request@lists.utsouthwestern.edu> > You can reach the person managing the list at> histonet-owner@lists.utsouthwestern.edu> > When replying, please edit your Subject line so it is more specific> than "Re: Contents of Histonet digest..."> > > Today's Topics:> > 1. Re: Histonet Digest, Vol 19, Issue 43 (Amos Brooks)> > > ----------------------------------------------------------------------> > Message: 1> Date: Sat, 02 Jul 2005 16:19:45 -0400> From: Amos Brooks > Subject: [Histonet] Re: Histonet Digest, Vol 19, Issue 43> To: histonet@lists.utsouthwestern.edu> Message-ID: <42C6F6E1.2010501@earthlink.net>> Content-Type: text/plain; charset=ISO-8859-1; format=flowed> > Carla,> > We use both depending on the antibody. Mostly we use Envision, but> we tried the Impress on some of the antibodies that don't label very> intensely and did see improvement. If you compare the results of a CD2,> CD4, CD7 or CD8 odds are you'll see some improvement. Not all the> antibodies showed marked improvement and in some cases envision was better> so we use Envision on most but the success we've had with Impress made it> worth switching some of the procedures. Vector might give you a sample if> you ask nicely. Give it a whirl and see if you like it.> > Best of luck,> Amos> > > > Message: 17> Date: Thu, 30 Jun 2005 07:34:30 -0700> From: Carla M Conway > Subject: [Histonet] Dako EnVision G2 or Vector ImmPRESS comments> wanted > To: Histonet@lists.utsouthwestern.edu> Message-ID:> > Content-Type: text/plain; charset=US-ASCII> > Hello all,> > I would like any comments (pro and con) regarding Dako's EnVision G2/AP> system or Vector's ImmPRESS kit. We are currently using EnVision + , but> are wondering if these new systems could be even better (more sensitive).> > Thanks for your help,> > Carla Conway> > Western Fisheries Research Center> 6505 NE 65th St> Seattle, WA 98115> ph: 206-526-6282 ext. 242> fax: 206-526-6654> cmconway@usgs.gov> > > > > > ------------------------------> > _______________________________________________> Histonet mailing list> Histonet@lists.utsouthwestern.edu> http://lists.utsouthwestern.edu/mailman/listinfo/histonet> > End of Histonet Digest, Vol 20, Issue 3> ***************************************> > This information is confidential and intended solely for the use of theindividual or entity to whom it is addressed. If you have received this email in error please notify the sender or ourCustomer Support Center at (262) 928-2777. We have scanned this email and its attachments for malicious content.However, the recipient should check this email and any attachments for thepresence of viruses.ProHealth Care accepts no liability for any damage caused by any virustransmitted by this email. ------------------------------Message: 12Date: Tue, 5 Jul 2005 10:39:33 -0500From: "Till, Renee" Subject: [Histonet] liver cryosectionsTo: histonet@lists.utsouthwestern.eduMessage-ID: Content-Type: text/plain; charset=us-asciiCan anyone offer some advice on cutting liver cryosections? My everyattempt produces shredded tissues. I know in paraffin embedding thatmeans they are dry and I would soak it in ice water before I cut, butwhat do you do with cryosections? I'm pretty sure it's not my cuttingtechnique, though I am fairly new at it. I've adjusted the roll plateand the vacume window. And I've tried it without the roll plate, thoughas we are just starting out with our cryostat I don't have any goodbrushes for pulling the section. Any ideas? Maybe they did not haveenough time to come down from -80 to -20? I left them in for about 2hours. Renee' ------------------------------Message: 13Date: Tue, 5 Jul 2005 11:45:01 -0400 From: "Monfils, Paul" Subject: RE: [Histonet] liver cryosectionsTo: "'histonet@lists.utsouthwestern.edu'" Message-ID: <09C945920A6B654199F7A58A1D7D1FDE01717565@lsexch.lsmaster.lifespan.org> Content-Type: text/plain; charset="ISO-8859-1"The block is too cold. -20 degrees is an appropriate temperature forsectioning many tissues, but some tissues require a lower temperature andothers require a higher temperature. For liver, try -15 degrees. For somesamples you may have to go as high as -12 degrees.Paul M.> ----------> From: histonet-bounces@lists.utsouthwestern.edu on behalf of Till,> Renee> Sent: Tuesday, July 5, 2005 8:39 AM> To: histonet@lists.utsouthwestern.edu> Subject: [Histonet] liver cryosections> > Can anyone offer some advice on cutting liver cryosections? My every> attempt produces shredded tissues. I know in paraffin embedding that> means they are dry and I would soak it in ice water before I cut, but> what do you do with cryosections? I'm pretty sure it's not my cutting> technique, though I am fairly new at it. I've adjusted the roll plate> and the vacume window. And I've tried it without the roll plate, though> as we are just starting out with our cryostat I don't have any good> brushes for pulling the section. Any ideas? Maybe they did not have> enough time to come down from -80 to -20? I left them in for about 2> hours.> > > > Renee' > > > > _______________________________________________> Histonet mailing list> Histonet@lists.utsouthwestern.edu> http://lists.utsouthwestern.edu/mailman/listinfo/histonet> > ------------------------------_______________________________________________Histonet mailing listHistonet@lists.utsouthwestern.eduhttp://lists.utsouthwestern.edu/mailman/listinfo/histonetEnd of Histonet Digest, Vol 20, Issue 5***************************************----------------------
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