RE: SPAM-LOW: [Histonet] RE: Staining with two primary antibodies from same host
I would add an FC block before the first primary, I use one from Innovex for 20-30 min before the serum blocks, it is expensive but worth it for a mouse on mouse detection system. Patsy Patsy Ruegg, HT(ASCP)QIHC IHCtech 12635 Montview Blvd. Ste.215 Aurora, CO 80045 720-859-4060 fax 720-859-4110 www.ihctech.net www.ihcrg.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of C.M. van der Loos Sent: Monday, September 07, 2009 1:36 AM To: anonwu...@gmail.com Cc: histonet@lists.utsouthwestern.edu Subject: SPAM-LOW: [Histonet] RE: Staining with two primary antibodies from same host Adam,Good point to prepare a protocol first, and then start staining! From theoretical point of view your protocol should work. However, I have tried this 'Jackson approach' using a Fab blocking step in a mouse-on-mouse situation (before the MOM kits were available) without success. There are at least two good solutions for double immunofluorescence using two primaries from the same species:Brouns et al. JHC 50:575-582, 2002 and Uchihara et al. JHC 51:1201-1206, 2003 applied a tyramide/fluorochrome detection for the first primary and a simple two-step for the second. Because the tyramide amplification is such a sensitive method, the first primary can be diluted up to a level that the second simple two-step detection cannot pick up signal from the first primary. Titration of the first primary antibody is most important in this procedure. You can in vitro label your goat primary with the Zenon (Invitrogen) kit based on anti-goat Fab fragments directly labeled with an Alexa fluorochrome. Next, you can built up a multistep indirect/direct double staining method:goat primary 1donkey anti-goat/fluorochrome 1normal goat serum (1:10) for blockinggoat primary 2-Zenon in vitro labeled with anti-goat Fab/fluorochrome 2lots of success with staining!ChrisChris van der Loos, PhD Dept. of Pathology Academic Medical Center M2-230 Meibergdreef 9 NL-1105 AZ Amsterdam The Netherlands Date: Fri, 4 Sep 2009 22:37:34 -0500 From: Adam . anonwu...@gmail.com Subject: [Histonet] Staining with two primary antibodies from same host To: histonet@lists.utsouthwestern.edu Hi all, I'm looking into staining with two primary antibodies from the same host, in this case goat. I've read a bit about this on Jackson Immunoresearch's website, but I wanted to run by my idea to get an idea if this is at all feasible. I want to stain mouse tissue with antigen X and antigen Y. I have a two polyclonal primaries: goat anti-mouse X and a goat anti-mouse Y. This is what I was thinking 1) Block in donkey serum for 1 hr at room temp. 2) Incubate with goat anti-mouse X overnight at 4C. Wash. 3) Incubate with Dylight 488 donkey anti-goat for 30 mins at room temp. Wash. 4) Reblock in donkey serum for 1 hr at room temp. 5) Incubate with 10 - 20 ug / mL unconjugated donkey anti-goat Fab for 1 hr at room temp -- cheapest I could find was at Rocklandhttp://www.rockland-inc.com/ccp8033-fab-fragment-of-affinity-purifi ed-anti-goat-igg-28-805-7102-805-7102.htm. Wash. 6) Incubate with goat anti-mouse Y overnight at 4C. Wash. 7) Incubate with biotin donkey anti-goat for 30 mins at room temp. Wash. 8) Incubate with avidin AMCA for 30 mins at room temp Would this work? Is there an easier or better way? What are the pitalls or tips you could offer? Hope you all aren't reading this during your long weekend, Adam ___ 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] CD33 antibody
I am having some trouble getting good staining with the CD33 antibody from Novocastra. I am trying to stain FFPE bone marrow biopsies decalcified in Formical for 1 hour prior to processing. The staining is very weak even at a 1:25 dilution using a pH 9.0 antigen retrieval solution (recommended) in a pressure cooker. Has anyone had any luck with this antibody on bone marrow? I have tried this protocol with the blood clots sent with the bone marrow biopsies and it works just fine so I am guessing that the decal is affecting the staining. Any thoughts?? Justin Peters, HTL (ASCP) IHC Supervisor Bostwick Laboratories(tm) For Absolute Confidence(r) 4355 Innslake Drive Glen Allen, Virginia 23060 Phone:(804) 967-9225 ext. 1831 Cell:(804) 822-6084 Email: jpet...@bostwicklaboratories.com mailto:jpet...@bostwicklaboratories.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Biocare Medical Antibodies
Hi All, Is anyone using the Biocare cocktails with their Ventana stainers? Particularly the ULTRA? What success or problems have you encountered? Thanks, Toni CONFIDENTIALITY NOTICE This message and any included attachments are from Somerset Medical Center and are intended only for the addressee. The information contained in this message is confidential and may contain privileged, confidential, proprietary and/or trade secret information entitled to protection and/or exemption from disclosure under applicable law. Unauthorized forwarding, printing, copying, distribution, or use of such information is strictly prohibited and may be unlawful. If you are not the addressee, please promptly delete this message and notify the sender of the delivery error by e-mail or you may call Somerset Medical Center's computer Help Desk at 908-685-2200, ext. 4050. -- Somerset Medical Center is the recipient of the 2009 Orthopedic Surgery Excellence Award(tm) from HealthGrades, the nation's leading health care ratings company. Visit Somerset Medical Center's Web site - www.somersetmedicalcenter.com - for news, event listings, health information and more. Join the Discussion: Facebook: www.somersetmedicalcenter.com/fb Twitter: www.twitter.com/SomersetMedCtr ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] RE: Staining with two primary antibodies from same host
Nice! I may try one or both of your procedures (Adam Andrea). ~Merced --On Monday, September 07, 2009 3:09 PM -0400 Andrea Hooper anh2...@med.cornell.edu wrote: I routinely stain with two primaries from same species. Mostly rat antibodies on mouse tissue. I have been fortunate enough so far not to have to do it in GFP containing tissue so I have utilized either commercially available FITc labeled primaries, or we label them ourselves. Then I use an anti-FITC Alexa 488 for boosting signal. Very nice results, some of which we have published. Here is the general outline of the protocol (email me if you want specifics): - Protein block - Unconjugated primary #1 antibody (for example rat anti-X) - Fluorophore conjugated secondary antibody (for example anti-rat IgG conjugated to CY3) - Block with Rat IgG in excess to block any sites on the secondary which could potentially bind to your second primary antibody. - FITC conjugated primary #2 (for example rat anti-Y-FITC labeled) - Alexa 488 conjugated anti-FITC X protein will be labeled red Y protein will be labeled green From: Adam . anonwu...@gmail.com Subject: [Histonet] Staining with two primary antibodies from same host To: histonet@lists.utsouthwestern.edu Hi all, I'm looking into staining with two primary antibodies from the same host, in this case goat. I've read a bit about this on Jackson Immunoresearch's website, but I wanted to run by my idea to get an idea if this is at all feasible. I want to stain mouse tissue with antigen X and antigen Y. I have a two polyclonal primaries: goat anti-mouse X and a goat anti-mouse Y. This is what I was thinking 1) Block in donkey serum for 1 hr at room temp. 2) Incubate with goat anti-mouse X overnight at 4C. Wash. 3) Incubate with Dylight 488 donkey anti-goat for 30 mins at room temp. Wash. 4) Reblock in donkey serum for 1 hr at room temp. 5) Incubate with 10 - 20 ug / mL unconjugated donkey anti-goat Fab for 1 hr at room temp -- cheapest I could find was at Rocklandhttp://www.rockland-inc.com/ccp8033-fab-fragment-of-affinity-pu rified-anti-goat-igg-28-805-7102-805-7102.htm. Wash. 6) Incubate with goat anti-mouse Y overnight at 4C. Wash. 7) Incubate with biotin donkey anti-goat for 30 mins at room temp. Wash. 8) Incubate with avidin AMCA for 30 mins at room temp Would this work? Is there an easier or better way? What are the pitalls or tips you could offer? Hope you all aren't reading this during your long weekend, Adam ___ 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 Merced M Leiker Research Technician II Cardiovascular Medicine 348 Biomedical Research Building State University of New York at Buffalo 3435 Main St, Buffalo, NY 14214 USA lei...@buffalo.edu 716-829-6118 (Ph) 716-829-2665 (Fx) No trees were harmed in the sending of this email. However, many electrons were severely inconvenienced. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] GMAblocks
Richard, I am assuming that you have already worked-up your antibodies on GMA embedded tissue.. If you have then you should be OK for months depending entirely on the epitope in question. If you have not then you could be in for an unpleasant surprise as many antibodies are not able to penetrate the polymerized GMA which cannot be etched. Robert Schoonhoven, HT/HTL (ASCP) From: Edwards, R.E. r...@leicester.ac.uk To: Histonet histonet@lists.utsouthwestern.edu Sent: Friday, September 4, 2009 11:29:29 AM Subject: [Histonet] GMAblocks Assuming that GMA blocks are stored under optimum conditions,i.e. with desiccant and at -20C; how long can they be stored and still obtain meaningful immunohistochemical staining?. Many thanks for any info Richard Edwards Leicester University...U.K. ___ 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] Dragon
We are currently researching the Dragon voice recognition system and I see that there is quite a bit of recent emails on the histonet concerning this subject. Is there anyone who is using Dragon that experienced reluctant Pathologists and how did you deal with that? I would be very interested in the mini steps you may have taken to go paperless and bring the Pathologists on board with this new idea. Thank you for sharing your experiences and suggestions with me. This histonet is such a great tool!!! Deanne Knutson Anatomic Pathology Supervisor St. Alexius Medical Center 900 E. Broadway Bismarck, North Dakota 58506 (701)-530-6730 dknut...@primecare.org mailto:dknut...@primecare.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] NSH One Day Forum in NJ
NSH is excited to announce a partnership with the New Jersey Society for Histotechnology to present a one day session dedicated to topics of interest for the histologist working in the research field. One low price includes continental breakfast and lunch. Sessions presented include: * The Use of In Situ Hybridization in Modern Drug Discovery, Dr. Paul Shughrue, Merck Research Laboratories Histologic Preparation of Decalcified Rat Incisors for Evaluating Compound Effects on Blood Vessel Density by IHC, Karen Phillips and Michele French, Bristol-Myers Squibb * Going LEAN in Research: 5S Visual Work Place, Carol Barone, Nemours - A. I. Dupont Hospital for Children * Insect Histology: Historical Overview and Current Perspectives, Damien Laudier, Laudier Histology * Troubleshooting IHC: Panel of Experts Panel Members: Maria Geraci-Erck, Schering-Plough, Tara Kennedy, Biocare Medical and Linda Watson, Bristol-Myers Squibb * Muscle Talk, Carol Barone, Nemours - A. I. Dupont Hospital for Children * A Histological Analysis of the Antiangiogenic Activity in Tumor Xenograft Models, Anne Lewin, Bristol-Myers Squibb For complete details visit www.nsh.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Dragon speech recognition
In my travels as a locum tenens pathologist, I have not seen Dragon or any other speech recognition system in use by pathologists, and can only recall one client who was even considering it. Computerized speech recognition could be disastrous for a small pathology practice, if management were to use its introduction as an excuse to fire the transcriptionist, who also answers the telephone and is the de facto practice administrator as well. Speech recognition systems depend on good microphones and on a quiet work area with minimal extraneous noise. Grossing stations are inherently noisy, and the working conditions (vibration, formaldehyde) quickly degrade microphones. Many pathologists' hospital offices are also very noisy. I'd like to get my hands on a speech recognition system, and I think I could learn it. My problem with it would be that I can type pathology reports about as fast as I can dictate them. Bob Richmond Samurai Pathologist Knoxville TN ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Job Opportunity - GS-0601-11 - Bethesda, MD
There is a full-time Federal civilian job opening at a DoD research facility (Armed Forces Radiobiology Research Institute) in Bethesda, Maryland. The position is for a Histotechnologist (Comparative Pathology) GS 0601-11 and was opened up for recruitment recently. The below link provides the full recruitment announcement out on the Department of the Navy (DON) CHART system. https://chart.donhr.navy.mil/jobsearch/jobdetailE.asp?vid=93997 Opening Number: NW9-0601-11-K3475832-I Opening Date: 9/5/09 Closing Date: 9/12/09 Steven R. Mog, DVM, Diplomate ACVP MAJ, VC, USA Chief, Comparative Pathology Division Veterinary Sciences Department Armed Forces Radiobiology Research Institute (AFRRI) Uniformed Services University of the Health Sciences (USUHS) Bethesda, MD 20889 301-295-1568 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Dragon speech recognition
Our pathologists' currently use dragon speech recognition. The newest version has worked out worlds better than the old system. Even our pathologist from India, with a fairly heavy accent, has happily used it. I must say that the system 5-6 years ago, did not meet their expectations and they all soon quit. But the latest one has worked out very well. Our pathologist's assistant however, chose not to use it this time, as the first time around was just a nightmare for him. And, as Dr. Richmond said below, maybe it was due to the noisy, smelly conditions. My non-typing husband uses a home-version to dictate letters to friends! He loves it! Robert Richmond rsrichm...@gmail.com 09/08/09 1:31 PM In my travels as a locum tenens pathologist, I have not seen Dragon or any other speech recognition system in use by pathologists, and can only recall one client who was even considering it. Computerized speech recognition could be disastrous for a small pathology practice, if management were to use its introduction as an excuse to fire the transcriptionist, who also answers the telephone and is the de facto practice administrator as well. Speech recognition systems depend on good microphones and on a quiet work area with minimal extraneous noise. Grossing stations are inherently noisy, and the working conditions (vibration, formaldehyde) quickly degrade microphones. Many pathologists' hospital offices are also very noisy. I'd like to get my hands on a speech recognition system, and I think I could learn it. My problem with it would be that I can type pathology reports about as fast as I can dictate them. Bob Richmond Samurai Pathologist Knoxville TN ___ 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] Re: Dragon speech recognition
Our two pathologists have just started using the Dragon speech recognition - the medical version that our hospital had purchased for other physicians. One of our pathologists, in trying out the software, read from a pathology textbook. It never missed a word. We have been using it for approximately three weeks now. It has eliminated a full time position (she had just given her notice one month ago). The other parts of the transcriptionist position have been absorbed by three laboratory secretaries. So far, the pathologists believe that using the Dragon software has decreased our turn around time. Lynne Bell, HT (ASCP) Central Vermont Medical Center 130 Fisher Road Berlin, VT 05602 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Dragon speech recognition
In my travels as a locum tenens pathologist, I have not seen Dragon or any other speech recognition system in use by pathologists, and can only recall one client who was even considering it. Computerized speech recognition could be disastrous for a small pathology practice, if management were to use its introduction as an excuse to fire the transcriptionist, who also answers the telephone and is the de facto practice administrator as well. Speech recognition systems depend on good microphones and on a quiet work area with minimal extraneous noise. Grossing stations are inherently noisy, and the working conditions (vibration, formaldehyde) quickly degrade microphones. Many pathologists' hospital offices are also very noisy. I'd like to get my hands on a speech recognition system, and I think I could learn it. My problem with it would be that I can type pathology reports about as fast as I can dictate them. Bob Richmond Samurai Pathologist Knoxville TN ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Pathologist Consultation Send out
Dr. Cartun's remarks describe the process followed at our facility. Vinnie Della Speranza Manager for Anatomic Pathology Services 165 Ashley Avenue Suite 309 Charleston, SC 29425 tel. 843-792-6353 fax. 843-792-8974 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Richard Cartun Sent: Sunday, September 06, 2009 10:59 AM To: histonet@lists.utsouthwestern.edu; rick.garnh...@memorialhealthsystem.com Subject: Re: [Histonet] Pathologist Consultation Send out Most pathologists that bill separately have a contract with the hospital to provide services. Therefore, in my opinion, if the pathologist can't provide a diagnosis and the case is sent out, the pathologist is responsible for the bill. However, if the case is sent out at the request of the patient or clinician (after the pathologist has established a diagnosis) then the patient (or her/his insurance) or the clinician is responsible for the bill. The hospital should not have to pay for these requests. Richard Richard W. Cartun, Ph.D. Director, Histology Immunopathology Director, Biospecimen Collection Programs Assistant Director, Anatomic Pathology Hartford Hospital 80 Seymour Street Hartford, CT 06102 (860) 545-1596 (860) 545-0174 Fax rick.garnh...@memorialhealthsystem.com 9/4/2009 1:07 PM If your Pathologists bill for the professional component privately, and your hospital bills for the technical component, who pay for expert consultations of slides sent out? Rick Garnhart HT(ASCP) Memorial Health System Histology Supervisor 1400 E. Boulder St. Colorado Springs, CO 80909 Cell: 719-365-8357 Ph: 719-365-6926 Fax: 719-365-6373 rick.garnh...@memorialhealthsystem.com Mission: To provide the highest quality health care Vision: To create an outstanding health system where patients heal and people thrive Values: Compassion - Integrity - Quality - Respect - Teamwork www.memorialhealthsystem.com The information contained in or attached to this electronic message is privileged and confidential, intended only for the use of the individual(s) named above. If the reader of this message is not the intended recipient, or the employee or agent responsible to deliver it to the intended recipient, you are hereby notified that any dissemination, distribution, or copying of this communication is strictly prohibited. If you have received this communication in error, please inform the sender immediately and remove any record of this message.___ 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
[Histonet] 1p/19q probe
I've been working on FISH on Her-2 neu and the neuro pathologist presumed I knew a protocol on Oligodendrogliomas. I was wondering if any one out there has used 1p 1q probe and if they have a protocol that they use on the Vysis 2000. I would really appreciate it. Rachel Pinch HT ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] New Histology Opening in Brooklyn, NY
New Histology position available in Brooklyn, New York. This is a full time permanent position with a privately held lab. It is a day shift and has the possibility of transitioning into supervisor role. Please call or email if interested Brian Feldman Principal Prometheus Healthcare Office 301-693-9057 Fax 301-368-2478 br...@prometheushealthcare.com mailto:br...@prometheushealthcare.com www.prometheushealthcare.com http://www.prometheushealthcare.com/ *** Stay up to date on the newest positions and healthcare trends nationwide on Twitter!*** http://twitter.com/PrometheusBlog ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
