[Histonet] Immunofluorescence double labeling with two rabbitantibodies

[Anatoli Gleiberman]

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: AGleiberman <@t> cbiolabs.com


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Aline
Rodrigues
Sent: Tuesday, October 12, 2010 1:29 PM
To: histonet <@t> 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 
    

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