[Histonet] RE: Acetone fixation problems with OCT Tissues
Patrick, We do a lot of frozen section IHC work. Years ago Gayle Callis turned me on to fixing in cold acetone:ethanol (3:1) . We keep it at -20C and I fix for 10 min. on the bench then wash in PBS and proceed with the IHC. We do dry slides for at least 30 min before fixing. This has worked well in our hands for many different antibodies. Brett Brett M. Connolly, Ph.D. Principle Scientist, Imaging Dept. Merck Co., Inc. PO Box 4, WP-44K West Point, PA 19486 brett_conno...@merck.com T- 215-652-2501 F- 215-993-6803 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Lewis, Patrick Sent: Tuesday, April 28, 2015 5:56 PM To: (Histonet@lists.utsouthwestern.edu) Subject: [Histonet] Acetone fixation problems with OCT Tissues Hi Everyone, I am still having issues with my IHCs with Acetone fixation. If I fix in 100% Acetone, I get IHC staining, but my tissues are 50-90% destroyed. If I fix in 4% paraformaldehyde, or 10% NBF or (95% Etoh and/or Methanol with Acetone) I lose the epitopes I either get no staining or very weak staining, but the tissue morphology look fine. I just tried an acetone gradient where I cut the tissues at 5 uM and dried them overnight, then fixed for 10 minutes in 100% acetone, then fixed in 95% acetone for 1 minute, then fixed in 70% acetone for 30 seconds, then quick rinsed in H20, then washed as normal in DPBS pH 7.4. I did 4 slides, 2 slides with one company's Charged slides ,and 2 slides with another company's charged slides. One company's slides look completely destroyed, the others may turn out, it was hard to tell how much damage there was. I'll know tomorrow when I finish staining and Hemotoxylin them. Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 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 protected by law. 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 Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (2000 Galloping Hill Road, Kenilworth, New Jersey, USA 07033), and/or its affiliates 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
RE: [Histonet] Re: Acetone fixation
We use acetone to denature fatty tumors without any problems. They sit in it for at least half an hour. I don't rinse them. Lynn M Burton Histology Animal Disease Lab Galesburg, Il 309-344-2451 -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Bob Richmond Sent: Saturday, November 15, 2014 2:34 PM To: Histonet@lists.utsouthwestern.edu Subject: [Histonet] Re: Acetone fixation Sheree H (where?) asks: Have questions about using acetone at the grossing counter. Does anyone have problems with it being carried over into the formalin on the processor? We rinse, open cassette to let it dry completely and even change the cassette. I've had issues with my processor and want to make sure it's not coming from the acetone. Acetone doesn't belong in the grossing area - too much of a fire and explosion hazard. If your pathologists are using it to fix marking ink onto specimens, they can use 3% acetic acid (half-strength white vinegar). I don't use anything at all - if you blot the specimen dry before you put the ink on, it'll stay put. Bob Richmond Samurai Pathologist Maryville 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
[Histonet] Re: Acetone fixation
Sheree H (where?) asks: Have questions about using acetone at the grossing counter. Does anyone have problems with it being carried over into the formalin on the processor? We rinse, open cassette to let it dry completely and even change the cassette. I've had issues with my processor and want to make sure it's not coming from the acetone. Acetone doesn't belong in the grossing area - too much of a fire and explosion hazard. If your pathologists are using it to fix marking ink onto specimens, they can use 3% acetic acid (half-strength white vinegar). I don't use anything at all - if you blot the specimen dry before you put the ink on, it'll stay put. Bob Richmond Samurai Pathologist Maryville TN ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Acetone Fixation
Atoska Gentry asked: hello, if any of you have acetone fixation incorporated into you frozen section H E staining protocol will you please advise me on adjustments necessary for routine staining protocol. Also, out of curiosity please enlighten me on the purpose for post sectioning acetone fixation on tissue samples initially fixed in 95% ETOH/ Acetic Acid? Your prompt replies will be greatly appreciated. ~ Atoska Several thoughts come to mind. Acetone fixation is usually used only for preserving antigenicity on fresh frozen tissue samples. For slides needing HE I would use a formalin fixative prior to the HE procedure for fresh frozen tissues. Acetone fixed HE stained cryosections are uuugly. You should not be doing frozen sections on tissues initially fixed in alcohol. Alcohol is an anti-freeze and you will not get good freezing/sectioning of the samples. You can section unfixed samples and then fix after mounting on glass slides in the Alcohol/acetic acid fix. Teri Johnson, HT(ASCP)QIHC Managing Director, Histology Facility Stowers Institute for Medical Research Kansas City, MO ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Acetone Fixation
hello, if any of you have acetone fixation incorporated into you frozen section H E staining protocol will you please advise me on adjustments necessary for routine staining protocol. Also, out of curiosity please enlighten me on the purpose for post sectioning acetone fixation on tissue samples initially fixed in 95% ETOH/ Acetic Acid? Your prompt replies will be greatly appreciated. ~ Atoska ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Acetone Fixation
What is your goal? A routine HE or immuno'setc.?? Atoska Gentry gent...@auburn.edu 3/25/2010 12:21 PM hello, if any of you have acetone fixation incorporated into you frozen section H E staining protocol will you please advise me on adjustments necessary for routine staining protocol. Also, out of curiosity please enlighten me on the purpose for post sectioning acetone fixation on tissue samples initially fixed in 95% ETOH/ Acetic Acid? Your prompt replies will be greatly appreciated. ~ Atoska ___ 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
