Hi Ross, Use sequential staining with monovalent anti-mouse Fab fragments (donkey or goat) from Jackson ImmunoResearch labeled with different fluorochromes - DyLight488 and DyLight594, for example. As blocking step I use 5% normal donkey serum. Usually it works fine.
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 Ross Benik Sent: Wednesday, January 26, 2011 6:47 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] dual fluorescent staining Hello everyone, I am working on a protocol for a dual fluorescent stain with two mouse antibodies (one is IgG1 and the other is IgG2a). We started by performing the two stains sequentially with a serum free protein blocking step in between, tagging one antibody with a 488 FITC labeled secondary and a 568 Cy3 secondary on the other (both goat anti mouse of course). With this method we are getting a lot of background staining so I was wondering if anyone who has experience in this sort of work if you have a recommendation of a proper blocking solution between the sequential stains? I've heard about using Rodent Block Rat from Biocare Medical or an un-conjugated goat anti mouse IgG secondary immunoglobulin but I would like to hear from you all as well! :-) Thanks, Ross _______________________________________________ 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
