[Histonet] FW: mimicry

[Sasa Jovanovic]

-----Original Message-----
From: Sasa Jovanovic [mailto:sasa <@t> jovanovic.co.za] 
Sent: 18 November 2005 07:05 AM
To: 'hstonet <@t> lists.utsouthwestern.edu'
Subject: FW: mimicry


 
-----Original Message-----
From: Sasa Jovanovic [mailto:sasa <@t> jovanovic.co.za] 
Sent: 17 November 2005 11:16 AM
To: 'Johnson, Teri'
Subject: mimicry


Dear Teri Johnson,
I am referring to your previous HIstonet correspondence  of Teri Johnson
where  they mentioned the following :....we also tried using an anti-IgG2a
anti mouse/HRP labelled secondary antibody, and in the negative control
there was no staining.....
I have red few of the Alon R papers regarding the mimicry between
streptavidin and fibronectin since I presume I  might have the same problem
In my project I am trying to determine presence/absence of alpha4beta 1
integrin in the rat uterus by using immunocytochemistry on the frozen
tissue....At the moment I have DAKO ARK kit and applying the following
procedure:
Cut 6um thick sections (use positive charged slides and silane pre-treated )
dry sections overnight at room temperature
fix them the next day in cold acetone for 10 minutes
proceed with staining:
Biotinylate primary antibody (monoclonal -mouse anti human CD49d (integrin
alpha 4 beta 1)P4G9.
Step 1: Mix diluent, concentrated primary Ab (positive or negative) and
biotinylation reagent, incubate for 15 min
Step 2: add blocking reagent incubate for 5 min
Apply peroxidase block for 15 min
Rinse
Apply prepared biotinylated primary Ab (positive or negative), incubate for
1 hour 
Rinse
Apply streptavidin HRP incubate for 15 min
Rinse
Apply prepared DAB incubate for 5 min
Rinse
Counterstain
Mount coverslips
 
I am using Tris-HCL pH9 (with added Tween 20)  as a washing buffer
I am using human tonsil as a positive control where I did not see any
specific immunolocalization on the sections where I had omit primary Ab.
But,
 
I am experiencing "staining" of my negative control, actually positive and
negative uterus look the same.....they "stain" on the same place.
Could this be the sterptavidin mimicry  and would you have any suggestions
how to incorporate anti IgG2a antibody in my kit or maybe do you have any
other solution to my problem?
You advice is greatly appreciated!!!
Thank you in advance
Sasha