[Histonet] RE: dual fluorescent staining

Anatoli Gleiberman AGleiberman <@t> cbiolabs.com
Thu Jan 27 09:20:43 CST 2011

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
e-mail: AGleiberman <@t> cbiolabs.com

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Ross Benik
Sent: Wednesday, January 26, 2011 6:47 PM
To: histonet <@t> 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! :-)    






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