[Histonet] Strepavidin blocking kit with your mimicry problem

[Gayle Callis]

Sasa,

Please read the following private communcation I had with Vector Technical 
services concerning the issue you just mentioned.

You may want to change your avidin/biotin blocking method to a 
Strepavidin/biotin blocking method (Vector kit) and use the DAKO kit with 
Strepavidin-HRP as it is intended with no secondary antibody involved.  I 
did not see any avidin nor Strepavidin/biotin blocking in your 
method.   Also, was your negative control the antibody isotype, which 
should be ARK'd the same as the primary and at the same concentration?
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Craig Pow (Vector) wrote:

Streptavidin does have some subtle binding differences than avidin. 
Streptavidin has been shown to bind to fibronectin receptors
that are expressed on cell types. These integrins (extracellular matrix 
receptors) mediate cell adhesion, migration etc and streptavidin binds to 
these too. So the bottomline this that streptavidin based detection systems 
may give background/non-specific staining due to this inetgrin binding that 
is not blocked by a standard avidin/biotin blocking step. Hence the idea
behind the streptavidin/biotin kit. See the following reference:

Alon, R. et al (1991) Cell-adhesive properties of streptavidin are mediated
by the exposure of an RGD-like RYD site. Eur. J. Cell Biol. 58:271-279.

The nomenclature in this article is dated. The GpIIbIIIa is now known as 
the alpha 5 beta 1 dimer that specifically binds fibronectin via the RGD 
amino acid sequence. There are antibodies available against the alpha 5 
subunit itself. Migratory cells, such as embryonic cells, some tumor cells 
etc would be high expressors of this integrin. Obviously you could either 
block with the streptavidin kit or use an avidin based detection system.

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At 10:07 PM 11/17/2005, you wrote:
>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?



Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)