Hi Carole, While I have no direct experience with human on human IHC, my first thought would be to biotinylate your primary antibody and then you would omit the anti-human secondary which is the most likely source of your background staining.
If you are using fluorescence you could also conjugate your primary with a fluorophore (http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/Zenon-Labeling-Technology.html) Brett M. Connolly, Ph.D. Principal 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 carole.chot...@fr.netgrs.com Sent: Friday, January 11, 2013 5:32 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] tricks about antibody generated in human on human tissue for IHC Dear all, I work in an histopathology department and we intend to do immunohistochemistry with an antibody generated in human on human tissues. So far, we don't have any experience with that. So I was wondering if somebody would have some feedback to prevent strong background generated by this human antibody on human tissue. We are planning in a first place to decrease the concentration of the secondary antibody Donkey@human which is recommended at 1/200 by Jackson, using it at 1/500 for example. But if anybody would have some experience with this kind of experiments, their tricks would be more than welcome. Best Regards, Carole Chotard Carole Chotard, Ph.D. Pharmacology Research Scientist Molecular Pharmacology and Pathophysiology Department IDRS 125 Chemin de Ronde 78290 Croissy-sur-Seine France Phone : +33 1 55 72 29 48 Fax : +33 1 55 72 25 96 carole.chot...@fr.netgrs.com _______________________________________________ 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. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), 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