Re: immuno on mouse tissue

From:Gayle Callis

I believe the ARK kit is based on biotinylation of a purified primary
antibody, not reacting it with the secondary antibody which effectively
eliminates a secondary antibody altogether.  The ARK kit biotinylated
primary is then detected with DAKO's Strepavidin-HRP conjugate and DAB or

One can purchase monoclonal antimouse primaries already biotinylated from
Pharmingen, Serotec, others, and is our favorite method!  We follow with an
in-house dilution of SA-HRP (any would work) and a favorite chromogen. 

On acetone/alcohol fixed frozen sections, our (Pharmingen) rat
antimouse-biotinylated CD4 (doesn't work with formalin fixed paraffin
embedded methods even with retrieval/digestions) 0.5mg/ml concentration
diluted to 1:15,000, with fading out at 1:20,000, followed by
Strepavidin-HRP, DAKO's DAB+ and DAB enhancer.  Glucose oxidase endogenous
peroxidase blocking is a must though or the DAB enhancer picks up the
tiniest amount of funky residual peroxidases/pseudoperoxidase in tissues.   

Just an example of using a biotinylated primary! More fun with murine IHC. 

However, in our case, crossreaction background is still a problem UNLESS
our biotinylated antibody is preceded by normal serum block containing some
mouse serum (we use 10% goat/2.5% mouse routinely, can vary) AND dilute the
biotinylated primary antibody IN this same normal serum block. ARK kits
also contain special blocks before using the biotinylated antibody. 

If, however, an antibody resists biotinylation with the ARK kit, and some
do, one may have to resort to a mouse to mouse staining kit, for mouse
monoclonal antibodies on mouse tissues.  Vector, Zymed, Innogenex, and
Scytek - the latter worked well in our lab, ??? other vendors - have
special kits for mouse to mouse IHC.  The Scytek kit has an excellent
blocking reagent plus we didn't have a purified mouse antimouse primary, we
had to use a biotinylated primary, which we substituted into the secondary
antibody step (secondary was eliminated!) proceeded with staining, and it
worked fine. 


 At 09:03 AM 3/5/02 -0700, you wrote:
>Some of the Immuno companies offer mouse on mouse detection systems.
>DAKO has something called ARK (Animal Research Kit) for this purpose.
>The basic concept is to react the antibody with the secondary in a test
>tube then apply it to the tissue avoiding the general non specific
>binding of the secondary to the tissue proteins.
>Patsy Ruegg

Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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