[Histonet] Methyl green going away...
Hi my boss requires me to counterstain some DAB immunohistochemistry with methyl green. I use for that purpose the methyl green in aquos solution 0.5% in 4.2 acetate buffer. The problem is that after the washes I dehydrate and the methyl green becomes extremely faint with the alcohols. My protocol requires long periods of alcoholic dehydration very graded. Is there any idea on how to save the methyl green. Can I use an alcoholic solution? Thank you very much. Alonso ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] CAP Question
I have a question regarding a new CAP checklist question dealing with ER/PR validation. The new question is ANP.22976 ER/PgR Validation and states: “If the laboratory performs immunohistochemistry for estrogen receptor (ER) and/or progesterone receptor (PgR) as a prognostic/predictive marker on breast carcinoma, the laboratory has documented appropriate validation for the assay(s). Note: Initial test validation should include a minimum of 40 cases (20 positive and 20 negative cases) for FDA-approved/cleared tests; laboratories should consider using higher numbers of test cases if a Laboratory Developed or Laboratory Modified Test is to be validated. Validation should be performed by comparing the laboratory’s results with another assay that has been appropriately validated. Acceptable concordance levels are 90% for positive results and 95% for negative results. If significant changes are made to the testing methods (e.g. antibody clones, antigen retrieval protocol or detection system) revalidation is required. “ Our original validation was done in 2005 and was performed with 20 cases. Do we need to revalidate to be compliant with this question? Should we start from scratch or just do an additional 20 cases? Any feedback is appreciated. Thanks! Schaundra Walton BS HTL(ASCP) Histology Supervisor ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] A big thanks
I would like to say a big thank you to everyone who replied and sent me ideas to get the wrinkles out of the diaphysis of the mouse tibias I was cutting. Will be trying out some of the tips I received. Thanks again Nancy Lowen ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] CAP Question -ER/PR Validation
You should pose that question to CAP via acc...@cap.org they answer quickly and when they do you can print out their answer and keep for when your inspectors come. When I asked them that question, they said that it was usually at the discretion of the pathologist in charge. But instead of taking my word for It, email them and when you receive their response, print it. Then follow their instructions. Dana Settembre -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Schaundra Walton Sent: Monday, April 16, 2012 11:34 AM To: Histonet Subject: [Histonet] CAP Question I have a question regarding a new CAP checklist question dealing with ER/PR validation. The new question is ANP.22976 ER/PgR Validation and states: If the laboratory performs immunohistochemistry for estrogen receptor (ER) and/or progesterone receptor (PgR) as a prognostic/predictive marker on breast carcinoma, the laboratory has documented appropriate validation for the assay(s). Note: Initial test validation should include a minimum of 40 cases (20 positive and 20 negative cases) for FDA-approved/cleared tests; laboratories should consider using higher numbers of test cases if a Laboratory Developed or Laboratory Modified Test is to be validated. Validation should be performed by comparing the laboratory's results with another assay that has been appropriately validated. Acceptable concordance levels are 90% for positive results and 95% for negative results. If significant changes are made to the testing methods (e.g. antibody clones, antigen retrieval protocol or detection system) revalidation is required. Our original validation was done in 2005 and was performed with 20 cases. Do we need to revalidate to be compliant with this question? Should we start from scratch or just do an additional 20 cases? Any feedback is appreciated. Thanks! Schaundra Walton BS HTL(ASCP) Histology Supervisor ___ 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] CAP Question
Shaundra, The CAP requirements are for those who are starting ER/PR for the first time, or changing antibodies to a new clone. If you did the validation years ago before those recommendations came out and have a history of running it successfully then you do not have to re-validate the procedure. If you change antibodies to new clones, then you would have to do the more extensive validation. Tim Morken -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Schaundra Walton Sent: Monday, April 16, 2012 8:34 AM To: Histonet Subject: [Histonet] CAP Question I have a question regarding a new CAP checklist question dealing with ER/PR validation. The new question is ANP.22976 ER/PgR Validation and states: “If the laboratory performs immunohistochemistry for estrogen receptor (ER) and/or progesterone receptor (PgR) as a prognostic/predictive marker on breast carcinoma, the laboratory has documented appropriate validation for the assay(s). Note: Initial test validation should include a minimum of 40 cases (20 positive and 20 negative cases) for FDA-approved/cleared tests; laboratories should consider using higher numbers of test cases if a Laboratory Developed or Laboratory Modified Test is to be validated. Validation should be performed by comparing the laboratory’s results with another assay that has been appropriately validated. Acceptable concordance levels are 90% for positive results and 95% for negative results. If significant changes are made to the testing methods (e.g. antibody clones, antigen retrieval protocol or detection system) revalidation is required. “ Our original validation was done in 2005 and was performed with 20 cases. Do we need to revalidate to be compliant with this question? Should we start from scratch or just do an additional 20 cases? Any feedback is appreciated. Thanks! Schaundra Walton BS HTL(ASCP) Histology Supervisor ___ 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] Re: Histonet Digest, Vol 101, Issue 21
Message: 1 Date: Mon, 16 Apr 2012 11:26:47 -0400 From: Alonso Martinez-Canabal alonso.martinezcana...@utoronto.ca Subject: [Histonet] Methyl green going away... To: histonet@lists.utsouthwestern.edu Message-ID: 000901cd1be5$5c48d470$14da7d50$@utoronto.ca Content-Type: text/plain; charset=US-ASCII Hi my boss requires me to counterstain some DAB immunohistochemistry with methyl green. I use for that purpose the methyl green in aquos solution 0.5% in 4.2 acetate buffer. The problem is that after the washes I dehydrate and the methyl green becomes extremely faint with the alcohols. My protocol requires long periods of alcoholic dehydration very graded. Is there any idea on how to save the methyl green. Can I use an alcoholic solution? Thank you very much. Alonso Alonso, Another alternative is; Drying the slide in the oven @~ 60c for 10 min after the methly green counterstain. Madeleine Huey BS, HTL/QIHC (ASCP) Supervisor - Pathology (IPOX Histology) madeleineh...@elcaminohospital.org ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Tissue Processor
Hi everyone, I was wondering if anyone had any input on the Logos tissue processor by Milestone. Is anyone currently using it and what are the pros and cons, etc. . Any information would be greatly appreciated. Thanks, Vicki Gauch AMCH Albany, NY ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: IHC in porcine tissues
Dear Colleagues What secondary antibody or polymer would be better to use for mouse primary antibody on FFPE porcine tissues?Also, please share your experience regarding macrophages detection in porcine arteries. I found couple in Abcam and Serotec (only for frozen), but your opinions is very valuable. Thank you Galina Deyneko Novartis, Cambridge, MA 617-782-1675 home 617-871-7613 w ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
RE: [Histonet] Re: IHC in porcine tissues
Galina Try MAC387 that is a macrophage/monocyte marker that cross reacts in porcine. We use the one from abcam. We have found that some of the anti-mouse polymer reagents cross react to porcine so we use a rabbit anti-mouse from Dako E0464 as a secondary and then an envision rabbit or strept avidin and that works much better. Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Manager Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308-1592 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell www.premierlab.com Ship to address: 1567 Skyway Drive, Unit E Longmont, CO 80504 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Galina Deyneko Sent: Monday, April 16, 2012 1:00 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Re: IHC in porcine tissues Dear Colleagues What secondary antibody or polymer would be better to use for mouse primary antibody on FFPE porcine tissues?Also, please share your experience regarding macrophages detection in porcine arteries. I found couple in Abcam and Serotec (only for frozen), but your opinions is very valuable. Thank you Galina Deyneko Novartis, Cambridge, MA 617-782-1675 home 617-871-7613 w ___ 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] Methyl green going away...
You could try dehydrating in acetone (very quick). Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 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, AUSTRALIA -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Alonso Martinez-Canabal Sent: Tuesday, 17 April 2012 1:27 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Methyl green going away... Hi my boss requires me to counterstain some DAB immunohistochemistry with methyl green. I use for that purpose the methyl green in aquos solution 0.5% in 4.2 acetate buffer. The problem is that after the washes I dehydrate and the methyl green becomes extremely faint with the alcohols. My protocol requires long periods of alcoholic dehydration very graded. Is there any idea on how to save the methyl green. Can I use an alcoholic solution? Thank you very much. Alonso ___ 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
