Aline, Jackson Immunoresearch provides protocol and reagents that are useful for solving your problem: http://www.jacksonimmuno.com/technical/fab-blok.asp I have used this approach to localize at the same sample simultaneously two different isoforms of a transcription factor with two different mouse monoclonals. Worked for me.
Anatoli Gleiberman, PhD Director of Histopathology Cleveland Biolabs, Inc 73 High Street Buffalo, NY 14203 phone:716-849-6810 ext.354 fax:716-849-6817 e-mail: agleiber...@cbiolabs.com -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Aline Rodrigues Sent: Tuesday, October 12, 2010 1:29 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Immunofluorescence double labeling with two rabbitantibodies Hello all, I using formalin fixed paraffin embedded specimens from fetuses from sheep to examine which cells are virally infected within the CNS. I am using IBA-1 to identify microglial cells. The IBA-1 antibody is a rabbit antibody and it works really well in my specimens. At the same time I am also using an antibody against the virus that I am studying that is also a rabbit Ab. For these samples I am using double labeling to determine if cells are infected or not. Both antibodies work, but I can use them together otherwise there is x- reaction. Does anyone has any suggestions of how to label tissues with two different antibodies that come from the same species. On immunoportal someone suggested to use 4%PFA to fix the tissues in between the first (primary and secondary Ab) and the second (primary and secondary Ab), however it did not work. PFA did not block the previously bound Abs. The other option that I would have would be to use IBA-1 from goat. My problem with this antibody coming from goat is that I can have x- reaction and moderate background since goat and sheep are very closely related. Please, if someone has any suggestions I would immensely appreciate! Thank you very much! I hope you have a wonderful day! Aline _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet This communication may contain privileged information. It is intended solely for the use of the addressee. If you are not the intended recipient, you are strictly prohibited from disclosing, copying, distributing or using any of this information. If you received this communication in error, please contact the sender immediately and destroy the material in its entirety, whether electronic or hard copy. This communication may contain nonpublic information about individuals and businesses subject to the restrictions of the Gramm-Leach-Bliley Act. You may not directly or indirectly reuse or redisclose such information for any purpose other than to provide the services for which you are receiving the information. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet