[Histonet] Antigen retrieval
Thanks for the input on ouor antigen retrieval problem. Biocare did a little troubleshooting for us, and things look great, at least for today. Is your lab playing the lottery? Our is: http://www.chicagonow.com/downsize-maybe/2016/01/sorry-gang-we-might-not-win-the-lottery-tonight/ Lester J. Raff, MD MBA UroPartners Medical Director Of Laboratory 2225 Enterprise Dr. Suite 2511 Westchester, Il 60154 Tel: 708-486-0076 Fax: 708-492-0203 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Antigen retrieval question. and a bit more
Hello 'netters, hope you all had a good weekend. We are having some difficulties with the pressure cooker chambers we use for antigen retrieval. What different methodologies would be recommended? If we went to a different methodology for retrieval, would each antigen have to be revalidated? How many slides/runs would you recommend? Thanks. For any interested, new blog post http://www.chicagonow.com/downsize-maybe/2016/01/why-did-my-cat-dump-me/ Lester J. Raff, MD MBA UroPartners Medical Director Of Laboratory 2225 Enterprise Dr. Suite 2511 Westchester, Il 60154 Tel: 708-486-0076 Fax: 708-492-0203 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Antigen retrieval survey
Your 2 minutes would be better spent looking in an immunohistochemistry textbook. A small but excellent one is Polak, J.M. and Van Noorden, S. (1997). Introduction to Immunocytochemistry, 2nd ed. Royal Microscopical Society Microscopy Handbooks, 37. Oxford: BIOS Scientific Publications. You will find that there is an optimal technique of antigen retrieval for each antigen that has been critically studied. Some conditions (such as pH6, close to 100C for an hour) are OK for many antigens. Some require more alkaline solutions (eg pH9, more section losses!) and a few respond best to heating in a more acid (eg pH2) solution. With lower temperatures (eg 80C) longer times are generally needed. All sorts of chemicals have been included in antigen retrieval solutions, often without obvious reasons or explanations. There are published papers that compare retrieval conditions for antigens of importance in diagnostic pathology. Retrieval can sometimes be achieved without heating, as with proteolytic enzymes or 3M urea. With a survey you may find out which antigen retrieval methods are used by most of those who reply, but you will not learn anything about how to choose and use the methods, or why their discovery about 25 years ago was an important technological advance. Check out this classic paper with Web of Science, Scopus, or Google Scholar: Shi, S.-R., Key, M.E. and Kalra, K.L. (1991). Antigen retrieval in formalin-fixed, paraffin-embedded tissue: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. Journal of Histochemistry and Cytochemistry 39:741-748. The PDF can be downloaded for free. This paper has been cited by thousands of other publications. The titles of recent citing articles may help you find a good retrieval procedure for the antigen that you need to detect immunohistochemically. John Kiernan UWO, London, Canada = = = On 23/06/15, Craig wrote: > Hi, > > I am conducting a short 2 min survey for my science/business class > examining current trends for antigen retrieval also known as heat induce > epitope retrieval. Response will be greatly appreciated! > > https://www.surveymonkey.com/s/7989LKR > > Best, > Craig Vollert > Graduate Student > Department of Pharmacological & Pharmaceutical Sciences > SR2 521B > College of Pharmacy > University of Houston > ___ > 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] Antigen retrieval survey
Hi, I am conducting a short 2 min survey for my science/business class examining current trends for antigen retrieval also known as heat induce epitope retrieval. Response will be greatly appreciated! https://www.surveymonkey.com/s/7989LKR Best, Craig Vollert Graduate Student Department of Pharmacological & Pharmaceutical Sciences SR2 521B College of Pharmacy University of Houston ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] antigen retrieval question
Lynn, I use the Decloaker and also stain for BVD, so will get in contact with you later this afternoon. Jan Shivers University of Minnesota Veterinary Diagnostic Lab St. Paul, MN 55108 On Thu, May 30, 2013 at 9:54 AM, Burton, Lynn wrote: > If anyone out there is using a decloaker for antigen retrieval, when doing > ihc staining, would you please contact me on my e-mail address? I have been > struggling with a BVD test for months. It is not working consistently and I > want to explore using this method along with our Benchmark XT stainer from > Ventana. > Thank you, > Lynn Burton > Galesburg Animal Disease Lab > Galesburg, Il > lynn.bur...@illinois.gov > ___ > 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
Re: [Histonet] antigen retrieval question
Is this with an older tissue sample? We don't do antigen retrieval, but we do have a proprietary technique for tissue restoration which can help with problematic IHC studies, especially in older FFPE tissue samples. See here for some examples: http://hematoxylin-eosin-tales.blogspot.com/2013/04/new-technique-for-improving-staining-in.html We could treat your sections for free if you like. Let me know if you are interested. Rob Day. On May 30, 2013, at 10:54 AM, "Burton, Lynn" wrote: > If anyone out there is using a decloaker for antigen retrieval, when doing > ihc staining, would you please contact me on my e-mail address? I have been > struggling with a BVD test for months. It is not working consistently and I > want to explore using this method along with our Benchmark XT stainer from > Ventana. > Thank you, > Lynn Burton > Galesburg Animal Disease Lab > Galesburg, Il > lynn.bur...@illinois.gov > ___ > 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] antigen retrieval question
If anyone out there is using a decloaker for antigen retrieval, when doing ihc staining, would you please contact me on my e-mail address? I have been struggling with a BVD test for months. It is not working consistently and I want to explore using this method along with our Benchmark XT stainer from Ventana. Thank you, Lynn Burton Galesburg Animal Disease Lab Galesburg, Il lynn.bur...@illinois.gov ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Antigen retrieval
Does anyone have a favorite antigen retrieval method for FFPE mouse tissues that they would be willing to share? I have been using citrate buffer Ph6.0 with poor to moderate results. Thanks for any help! Mike ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] antigen retrieval
To those with the Biocare intelliPATH system, Is the antigen retrieval part of the IHC automated or do you need to use the Biocare decloaker? Thankyou, Tom Truscott ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Antigen retrieval survey
96 degree water bath for half an hour in either citrate buffer (pH 6) or Tris-HCl (pH 9), depending on which gives better retrieval for that antibody. -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea T. Hooper Sent: Wednesday, July 13, 2011 11:10 PM To: Histonet Subject: [Histonet] Antigen retrieval survey Hi All, I am doing a survey and will be happy to compile results and share if folks will respond! What is your favorite antigen retrieval method and/or panel? Buffer Source/composition Temperature Device Thanks, Andrea ___ 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] Antigen Retrieval
Citrate buffer is my first port of call. Jim Reilly ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Antigen retrieval survey
I hate to be pessimistic, but different clones of monoclonal antibodies can have different optimum retrieval requirements, not to mention tissue type, source species, etc. What I do believe however, is that enzymatic retrieval is under-sold - no equipment purchase is necessary so you will not see companies advocating their use. Nearly half of the antibodies we use are optimally retrieved with enzymes (pronase, trypsin, pepsin) rather than heat retrieval. Tresa Goins Veterinary Diagnostic Lab South 19th and Lincoln Bozeman, MT 59718 406-994-6353 - phone 406-994-6344 - fax Tresa -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Andrea T. Hooper Sent: Wednesday, July 13, 2011 9:10 PM To: Histonet Subject: [Histonet] Antigen retrieval survey Hi All, I am doing a survey and will be happy to compile results and share if folks will respond! What is your favorite antigen retrieval method and/or panel? Buffer Source/composition Temperature Device Thanks, Andrea ___ 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
Re: [Histonet] Antigen retrieval survey
Citrate buffer René J. --- On Wed, 7/13/11, Andrea T. Hooper wrote: From: Andrea T. Hooper Subject: [Histonet] Antigen retrieval survey To: "Histonet" Date: Wednesday, July 13, 2011, 11:10 PM Hi All, I am doing a survey and will be happy to compile results and share if folks will respond! What is your favorite antigen retrieval method and/or panel? Buffer Source/composition Temperature Device Thanks, Andrea ___ 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] Antigen retrieval survey
Hi All, I am doing a survey and will be happy to compile results and share if folks will respond! What is your favorite antigen retrieval method and/or panel? Buffer Source/composition Temperature Device Thanks, Andrea ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Antigen Retrieval for 10u sections in IHC
Hi tistonetters, I have to do IHC on 10µ sections. Is procedure for Antigen Retrieval same like for 4-5µ (time and temperature) Thanks in advance, Naira ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Antigen retrieval and myosin heavy chain question
Dear Histonetters, I was wondering if anyone out there has used antigen retrieval methods to facilitate immuno-staining of formalin-fixed skeletal muscle fibers? I am using ALD-58 + ABC Elite Mouse kit to stain slow-tonic fibers in fresh amphibian muscles, but need to take a look at the fiber distribution of some rare museum specimens. Any suggestions are welcome. Thanks! -Mike -- Mike Jorgensen PhD Candidate Department of Biological Sciences Ohio University Athens, Ohio 45701 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Antigen retrieval question
Consider the following sequence:
1- the NBF corsslinked the antigens making them "more" stable, "insolubable".
2- you did HIER and that crosslinkage disappeared, making the antigen
"vulnerable" or able to be dissolved
3- you left them in buffer while "vulnerable". Does not sound to you as if the
antigens were "diluted", "dissolved" in the buffer causing a weaker reaction
than usual?
That would be my explanation, but to your original question ("Have you
experienced...") my answer is no, I always proceeded with the IHC test
immediately after HIER. Maybe that is why it has not happened to me.
Just a thought!
René J.
--- On Tue, 10/20/09, Connolly, Brett M wrote:
From: Connolly, Brett M
Subject: [Histonet] Antigen retrieval question
To: histonet@lists.utsouthwestern.edu
Date: Tuesday, October 20, 2009, 2:21 PM
Has anyone experienced a "reversal" of citrate HEIR unmasking do to a
prolonged delay in continuing the experiment after the retrieval step?
Brett M. Connolly, Ph.D.
Research Fellow, Imaging Dept.
Merck & Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
tel. 215-652-2501 fax. 215-993-6803
brett_conno...@merck.com
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MSD and in Japan, as Banyu - direct contact information for affiliates is
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Re: [Histonet] Antigen retrieval question
Sounds like the retrieved antigen was lost rather than "reversal" Janet QIHC/HTL --- On Tue, 10/20/09, Connolly, Brett M wrote: From: Connolly, Brett M Subject: [Histonet] Antigen retrieval question To: histonet@lists.utsouthwestern.edu Date: Tuesday, October 20, 2009, 11:21 AM Has anyone experienced a "reversal" of citrate HEIR unmasking do to a prolonged delay in continuing the experiment after the retrieval step? Brett M. Connolly, Ph.D. Research Fellow, Imaging Dept. Merck & Co., Inc. PO Box 4, WP-44K West Point, PA 19486 tel. 215-652-2501 fax. 215-993-6803 brett_conno...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ 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] Antigen retrieval question
Has anyone experienced a "reversal" of citrate HEIR unmasking do to a prolonged delay in continuing the experiment after the retrieval step? Brett M. Connolly, Ph.D. Research Fellow, Imaging Dept. Merck & Co., Inc. PO Box 4, WP-44K West Point, PA 19486 tel. 215-652-2501 fax. 215-993-6803 brett_conno...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[HISTONET] Antigen retrieval paraffin skin samples
Dear all, Might anyone be able to offer their advice on the best method to retrieve antigens on paraffin embedded skin sections? I am immunostaining for epithelial cytokeratins, and I have block-to-block variation in the quality of the staining - some block give consistently good staining while others giving negligible staining. The tissues were prepared by fixation in 4% PFA for 24 - 48 hours followed by dehydration and embedding. I currently use 15 mins Ficin digestion. I suspect that the differences may be accounted for by variability in the length of time samples were fixed in PFA and then 70% ethanol. Best wishes Nick ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Antigen retrieval
I am attempting to do IF on FFPE sections but the history/treatment of my tissues (mouse tissues) is unknown. I would like to recommend to our labs a strategy for tissue collection, length of time in NBF, type of antigen retrieval, and so on. Would anyone (or everyone) be willing to share their opinions on the best methods they have for maintaining the ability to do IF or IHC. Thanks for any help! Mike ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] antigen retrieval-pressure cooker-CD44- CD24-canine tissue
Anjan,
I am more of a fan of steamer or waterbath than pressure cooker, in
my= hands at altitude the pressure cooker can be very harsh and make
my tissue= s fall off the slide or get chewed up badly. I know the
pressure is s= upposed to be controled by the cooker but in my hands
boiling seems to be t= aking place, violent boiling and i do not like
it.
are you sure the antibodies work on canine tissue? When testingabs,
I have four protocols I run
1. no pretreatment
2. HIER with lph citrate buffer
3. HIER with hph buffer, such as edta
4. EIER, I will start with proteinase K for 5min.
usually with one of these, if the ab x reacts with canine tissue you
w= ould get some signal, if it is weak i would increase the
pretreatment times= for that one approach. If i get nothing from any
of them and I know = I am using the optomal dilution of the primary ab
for the longest incubatio= n time (sometimes i go to over night
incubation of primary) then I really w= onder if that ab works on the
species I am trying to use it on.
Cheers,
Patsy
Original Message
Subject: [Histonet] antigen r= etrieval-pressure cooker-CD44-
CD24-canine
tissue
From: anjan kumar &= lt;drvet_an...@hotmail.com>
Date: Sun, May 24, 2009 1:48 pm
To: tr= iple immunohistochem
h= ello everyone,
I wanted to know the exact stepwise protocol for antigen = retrieval
using a pressure cooker. i tried using some protocol but there is= no
staining. Kindly tell me the detailed protocol, like when i should
plac= e the slides into the pressure cooker and from when i should
count the time= .
And i am using mouse monoclonals CD44 & CD24 labvision in can= ine
tissue, has anyone got experience with these antibodies as citrate
buff= er doesnt at all seems to work.
plz send me some information on this.
Regards,
Dr. Anjan Kumar.K.R
M.V.Sc Scholar
Dept. of = Veterinary Pathology
Madras Veterinary College
Chennai-7
<= BR>India
email: drvet_an...@hotmail.com
Phone: +91-9940475801
___= __
Live Search extreme As India feels the heat of poll season, get a= ll
the info you need on the MSN News Aggregator
[1]http://news.in.msn.com/National/indiael
ections2009/aggregator/default.aspx___
_= ___
Histonet mailing list
histo...@lists.utsouthwestern.edu[2]http://lists.utsouthwestern.edu/ma
ilman/listinfo/histonet
<= /DIV>
References
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2. 3D"http://lists.utsouthwestern.edu/mailman/listinfo/histonet";
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[Histonet] antigen retrieval-pressure cooker-CD44- CD24-canine tissue
hello everyone, I wanted to know the exact stepwise protocol for antigen retrieval using a pressure cooker. i tried using some protocol but there is no staining. Kindly tell me the detailed protocol, like when i should place the slides into the pressure cooker and from when i should count the time. And i am using mouse monoclonals CD44 & CD24 labvision in canine tissue, has anyone got experience with these antibodies as citrate buffer doesnt at all seems to work. plz send me some information on this. Regards, Dr. Anjan Kumar.K.R M.V.Sc Scholar Dept. of Veterinary Pathology Madras Veterinary College Chennai-7 India email: drvet_an...@hotmail.com Phone: +91-9940475801 _ Live Search extreme As India feels the heat of poll season, get all the info you need on the MSN News Aggregator http://news.in.msn.com/National/indiaelections2009/aggregator/default.aspx___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Antigen Retrieval on MMA slides
We put our plastic sections (5-6 microns in thickness) onto slides manufactured by Scientific Device Laboratories, the "in situ plus" slides. We then bake the sections overnight in a 60 degree oven, then bring to room temperature before doing immunostaining. Sections will not stay on gel coated slides and the gelatin on the slides will interfere with your immunostaining. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: RE: [Histonet] Antigen Retrieval
Hi, adjust the gelatin concentration! Our experiences of brain section (95 degree boiling for 30 min): 0.5% gelatin-coated slides coming off sections, not for 0.25%. Also, try to use "floating sections" rather direct attach frozen sections embedded with O.C.T on to slides. 2008-10-06 tf 发件人： Swain, Frances L 发送时间： 2008-10-06 19:44:38 收件人： Sebree Linda A.; Patten, Nicole (NIH/NIAAA) [F]; histonet@lists.utsouthwestern.edu 抄送： 主题： RE: [Histonet] Antigen Retrieval I work with non-decalcified and decalcified bone. After I have tried to pressure cook it, steam it, microwave it, etc my sections look pretty bad(coming off, etc.) I have found that either using Pepsin (Zymed) for 20 minutes at 37 degrees C. in a humidifying chamber or Pepsin for 20 minutes at room temperature in a humidifying chamber or microwave the buffer to boiling, quickly remove and place slides in solution, cover and let sit for 20 minutes or until cool to touch work the best for my purposes. Frances L. Swain HT(ASCP) A. A. S. Special Procedures Technician Department of Orthopaedic Surgery Center for Orthopaedic Research Barton Research Building 2R28 4301 West Markham Street Little Rock AR 72205 (501) 686-8739 PHONE (501) 686-8987 FAX [EMAIL PROTECTED] email -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Sebree Linda A. Sent: Friday, October 03, 2008 12:49 PM To: Patten, Nicole (NIH/NIAAA) [F]; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Antigen Retrieval Try using a laboratory pressure cooker like the Decloaking Chamber from Biocare Medical. Linda A. Sebree University of Wisconsin Hospital & Clinics IHC/ISH Laboratory DB1-223 VAH 600 Highland Ave. Madison, WI 53792 (608)265-6596 -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Patten, Nicole (NIH/NIAAA) [F] Sent: Friday, October 03, 2008 12:28 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Antigen Retrieval Hi- I am new to using IHC and I need some advice on Antigen Retrieval. I usually do it in an autoclave for 15min at 121C for FFPE human brain tissue (in Citrate or Tris Buffer), but by the end my tissue looks pretty bad and parts have even fallen off. The tissue is fixed on SuperFrost Plus Slides. Any suggestions would be really helpful. Thanks! N. Patten Post-Baccalaureate Fellow/IRTA National Institutes of Health ___ 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 Confidentiality Notice: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Antigen Retrieval
I work with non-decalcified and decalcified bone. After I have tried to pressure cook it, steam it, microwave it, etc my sections look pretty bad(coming off, etc.) I have found that either using Pepsin (Zymed) for 20 minutes at 37 degrees C. in a humidifying chamber or Pepsin for 20 minutes at room temperature in a humidifying chamber or microwave the buffer to boiling, quickly remove and place slides in solution, cover and let sit for 20 minutes or until cool to touch work the best for my purposes. Frances L. Swain HT(ASCP) A. A. S. Special Procedures Technician Department of Orthopaedic Surgery Center for Orthopaedic Research Barton Research Building 2R28 4301 West Markham Street Little Rock AR 72205 (501) 686-8739 PHONE (501) 686-8987 FAX [EMAIL PROTECTED] email -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Sebree Linda A. Sent: Friday, October 03, 2008 12:49 PM To: Patten, Nicole (NIH/NIAAA) [F]; histonet@lists.utsouthwestern.edu Subject: RE: [Histonet] Antigen Retrieval Try using a laboratory pressure cooker like the Decloaking Chamber from Biocare Medical. Linda A. Sebree University of Wisconsin Hospital & Clinics IHC/ISH Laboratory DB1-223 VAH 600 Highland Ave. Madison, WI 53792 (608)265-6596 -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Patten, Nicole (NIH/NIAAA) [F] Sent: Friday, October 03, 2008 12:28 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Antigen Retrieval Hi- I am new to using IHC and I need some advice on Antigen Retrieval. I usually do it in an autoclave for 15min at 121C for FFPE human brain tissue (in Citrate or Tris Buffer), but by the end my tissue looks pretty bad and parts have even fallen off. The tissue is fixed on SuperFrost Plus Slides. Any suggestions would be really helpful. Thanks! N. Patten Post-Baccalaureate Fellow/IRTA National Institutes of Health ___ 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 Confidentiality Notice: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Antigen Retrieval
Nicole, You might look into using a more gentle heat source, such as a vegetable steamer or water bath. There are also pressure cookers for IHC which may also be easier on your tissue that the autoclave, although I have had problems with them as well and stick to steamer or waterbath myself. Patsy Patsy Ruegg, HT(ASCP)QIHC IHCtech 12635 Montview Blvd. #215 Aurora, CO 80045 720-859-4060 fax 720-859-4110 [EMAIL PROTECTED] www.ihctech.net www.ihcrg.org -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Patten, Nicole (NIH/NIAAA) [F] Sent: Friday, October 03, 2008 11:28 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Antigen Retrieval Hi- I am new to using IHC and I need some advice on Antigen Retrieval. I usually do it in an autoclave for 15min at 121C for FFPE human brain tissue (in Citrate or Tris Buffer), but by the end my tissue looks pretty bad and parts have even fallen off. The tissue is fixed on SuperFrost Plus Slides. Any suggestions would be really helpful. Thanks! N. Patten Post-Baccalaureate Fellow/IRTA National Institutes of Health ___ 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
RE: [Histonet] Antigen Retrieval
Try using a laboratory pressure cooker like the Decloaking Chamber from Biocare Medical. Linda A. Sebree University of Wisconsin Hospital & Clinics IHC/ISH Laboratory DB1-223 VAH 600 Highland Ave. Madison, WI 53792 (608)265-6596 -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Patten, Nicole (NIH/NIAAA) [F] Sent: Friday, October 03, 2008 12:28 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Antigen Retrieval Hi- I am new to using IHC and I need some advice on Antigen Retrieval. I usually do it in an autoclave for 15min at 121C for FFPE human brain tissue (in Citrate or Tris Buffer), but by the end my tissue looks pretty bad and parts have even fallen off. The tissue is fixed on SuperFrost Plus Slides. Any suggestions would be really helpful. Thanks! N. Patten Post-Baccalaureate Fellow/IRTA National Institutes of Health ___ 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] Antigen Retrieval
Hi- I am new to using IHC and I need some advice on Antigen Retrieval. I usually do it in an autoclave for 15min at 121C for FFPE human brain tissue (in Citrate or Tris Buffer), but by the end my tissue looks pretty bad and parts have even fallen off. The tissue is fixed on SuperFrost Plus Slides. Any suggestions would be really helpful. Thanks! N. Patten Post-Baccalaureate Fellow/IRTA National Institutes of Health ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] antigen retrieval for 5 year pap smear slides
Are your PAP smear slides fixed in alcohol? If so, you probably don't need antigen retrieval. Richard Richard W. Cartun, Ph.D. Director, Histology & Immunopathology Assistant Director, Anatomic Pathology Hartford Hospital 80 Seymour Street Hartford, CT 06102 (860) 545-1596 (860) 545-0174 Fax >>> "hao zhang" <[EMAIL PROTECTED]> 09/13/08 11:11 PM >>> Hi, everyone I want to do IHC from a 5 year old pap smear slides for pan-keratin stain. I tried many methods of antigen retrieval protocols (10 mM citric buffer, ph 6; 1 mM EDTA, Ph8; 10 mM Tris buffer, Ph 10; ) and heat at 95C for 40 minutes. I also used Triton x 100 (0.2%) pretreatment 10 minutes and then SDS (0.5% or 0.25%) treatment for 10 minutes. But all method did not work. Any replies will be very appreciated. -- Hao ZHANG, Ph.D Scientist ___ 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
RE: [Histonet] antigen retrieval for 5 year pap smear slides
Hao,
It might be possible that not all of the mountant has been removed. Though I am
surprised that the staining is not better.
It just might be one of those instances where the keratin epitopes are damaged
during the 5 year storage.
Possibly the phosphotungstic acid, often included in the PAP stains, might also
be effecting the epitopes.
Again try these variables on buccal or sputum smears and compare the results.
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145
-Original Message-
From: hao zhang [mailto:[EMAIL PROTECTED]
Sent: Monday, 15 September 2008 12:22 PM
To: Tony Henwood
Subject: Re: [Histonet] antigen retrieval for 5 year pap smear slides
Hi, Tony, René and all,
I agree that pap smear is fixed by alcohol instead of formalin. Problem
for me now is that when I do IHC for pan-keratin stain I only find small part
cell stained (around 10%) even when I dont do antigen retrival by HIER. I know
it is around 5 year old slides but I dont know why I can not get higher
positive percentage because I used Hela cell as a control I got around 90%
positive percentage. In addition, I find a lot of pink color material remained
in the slide. I used 3% acid alcohol for destaining treatment and destaining
effect get improved but finally I still get less positive percentage for
pan-keratin. any suggest will be very appreciated.
On Sun, Sep 14, 2008 at 5:26 PM, Tony Henwood <[EMAIL PROTECTED]> wrote:
Do not bother to retrieve.
Usually PAP smears are not fixed in formalin but are alcohol
fixed.
Alcohol is an excellent fixative for intermediate filaments as
well as
keratins.
I would just de-coverslip the slides, rinse in buffer, block
endogenous
enzyme, add your diluted antibody (you will probably find that
you may
need to dilute your antibody further than for FFPE sections
(maybe up to
1-100 times more)) and finally demonstrate the bound antibody.
For titreing prepare some buccal smears, fix in the same
fixative used
for the PAP smears (95% ethanol or whatever commercial Cyto
spray fix
that is used), and work up the titreing as usual.
No antigen retrieval is required unless formalin cross-links
have been
formed.
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145
-Original Message-
From: [EMAIL PROTECTED]
[mailto:[EMAIL PROTECTED] On Behalf Of hao
zhang
Sent: Sunday, 14 September 2008 1:12 PM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] antigen retrieval for 5 year pap smear
slides
Hi, everyone
I want to do IHC from a 5 year old pap smear slides for
pan-keratin
stain.
I tried many methods of antigen retrieval protocols (10 mM
citric
buffer, ph 6; 1 mM EDTA, Ph8; 10 mM Tris buffer, Ph 10; ) and
heat at
95C for 40 minutes. I also used Triton x 100 (0.2%)
pretreatment 10
minutes and then SDS (0.5% or 0.25%) treatment for 10 minutes.
But all
method did not work. Any replies will be very appreciated.
--
Hao ZHANG, Ph.D
Scientist
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RE: [Histonet] antigen retrieval for 5 year pap smear slides
Do not bother to retrieve. Usually PAP smears are not fixed in formalin but are alcohol fixed. Alcohol is an excellent fixative for intermediate filaments as well as keratins. I would just de-coverslip the slides, rinse in buffer, block endogenous enzyme, add your diluted antibody (you will probably find that you may need to dilute your antibody further than for FFPE sections (maybe up to 1-100 times more)) and finally demonstrate the bound antibody. For titreing prepare some buccal smears, fix in the same fixative used for the PAP smears (95% ethanol or whatever commercial Cyto spray fix that is used), and work up the titreing as usual. No antigen retrieval is required unless formalin cross-links have been formed. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145 -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of hao zhang Sent: Sunday, 14 September 2008 1:12 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] antigen retrieval for 5 year pap smear slides Hi, everyone I want to do IHC from a 5 year old pap smear slides for pan-keratin stain. I tried many methods of antigen retrieval protocols (10 mM citric buffer, ph 6; 1 mM EDTA, Ph8; 10 mM Tris buffer, Ph 10; ) and heat at 95C for 40 minutes. I also used Triton x 100 (0.2%) pretreatment 10 minutes and then SDS (0.5% or 0.25%) treatment for 10 minutes. But all method did not work. Any replies will be very appreciated. -- Hao ZHANG, Ph.D Scientist ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet * This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are 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 The Children's Hospital at Westmead This note 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. ** ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] antigen retrieval for 5 year pap smear slides
Usually PAP smears are NOT fixed with NBF so they should NOT be treated with HIER before doing IHC. If you have some spare smear do IHC directly (after destaining). René J. --- On Sat, 9/13/08, hao zhang <[EMAIL PROTECTED]> wrote: From: hao zhang <[EMAIL PROTECTED]> Subject: [Histonet] antigen retrieval for 5 year pap smear slides To: histonet@lists.utsouthwestern.edu Date: Saturday, September 13, 2008, 11:11 PM Hi, everyone I want to do IHC from a 5 year old pap smear slides for pan-keratin stain. I tried many methods of antigen retrieval protocols (10 mM citric buffer, ph 6; 1 mM EDTA, Ph8; 10 mM Tris buffer, Ph 10; ) and heat at 95C for 40 minutes. I also used Triton x 100 (0.2%) pretreatment 10 minutes and then SDS (0.5% or 0.25%) treatment for 10 minutes. But all method did not work. Any replies will be very appreciated. -- Hao ZHANG, Ph.D Scientist ___ 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] antigen retrieval for 5 year pap smear slides
Hi, everyone I want to do IHC from a 5 year old pap smear slides for pan-keratin stain. I tried many methods of antigen retrieval protocols (10 mM citric buffer, ph 6; 1 mM EDTA, Ph8; 10 mM Tris buffer, Ph 10; ) and heat at 95C for 40 minutes. I also used Triton x 100 (0.2%) pretreatment 10 minutes and then SDS (0.5% or 0.25%) treatment for 10 minutes. But all method did not work. Any replies will be very appreciated. -- Hao ZHANG, Ph.D Scientist ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Antigen retrieval 'tween night and next day
Brian, I haven't seen your procedure for antigen retrieval before. I usually see heat/citrate buffer or some kind of protease treatment. It seems pretty straightforward. Have you ever used this procedure for keratins or basement membrane proteins? Brian wrote: I have performed antigen retrieval by leaving slides overnight in the refrigerator immersed in PBS + 0.05% Tween 20. I'm sure there are many other ways to accomplish antigen retrieval, heat merely accelerates the process. Thanks. Mel Melville B. Vaughan, Ph. D. Assistant Professor of Biology Campus Coordinator, NSF Sure-Step program University of Central Oklahoma 100 N. University Drive Edmond, OK 73034 Telephone 405-974-5725 Fax 405-974-5726 - **CONFIDENTIALITY** -This email (including any attachments) may contain confidential, proprietary and privileged information. Any unauthorized disclosure or use of this information is prohibited. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
