Hi Gayle,
Not very if there is minimal target.
With that endothelium being so very, very thin where that particular
cassette expresses, you can see it, barely, without TSA.  With TSA is
fantastic.
If wherever your b-gal is being expressed in your particular model, if it is
a nice big, plump cell you don't need amplification.   But if your promoter
and particular experiment means there is just not a lot of b-gal there in
first place, need the TSA.
For your particular experiment, if robust target there, amplification not
needed.  If cell type and or method of delivery makes target less robust,
the TSA works wonders.
Ray

-----Original Message-----
From: Gayle Callis [mailto:gcallis@montana.edu] 
Sent: Friday, December 08, 2006 10:00 AM
To: Koelling, Ray; Histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] GFP and B-Gal positive control slides

Ray,

A question: How effective is your antibody without having to use TSA 
amplification?

>Rebecca,
>
>The GFP antibodies we use are quirky so I don't want to make a
>recommendation.  At Jax where you can get the Tie-2/lacZ transgenic mice,
>you can also get GFP expressing mice for controls.  I'm not recommending,
>just a starting point for your search.  Look under research models on their
>web site.
>
>For the Tie-2/lac Z mice.  Fix in formalin and process to paraffin as
usual.
>We use an antibody from Cortex Biochem (San Leandro, CA) that is IgG
>fraction on rabbit polyclonal to beta-galactosidase.  Around 2-3 ug/ml,
>after a 5 min proteinase K digestion.  We use an amplification kit of the
>third step (TSA for 5 minutes).  DAB, etc.  The signal is outstanding.
>
>Ray
>
>Raymond Koelling
>Research Scientist
>Amgen Seattle
>
>-----Original Message-----
>From: histonet-bounces@lists.utsouthwestern.edu
>[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Rebecca
>Nishi
>Sent: Thursday, December 07, 2006 1:34 PM
>To: histonet@lists.utsouthwestern.edu
>Subject: [Histonet] GFP and B-Gal positive control slides
>
>Does anyone know where I can buy, or can suggest an easy way to make
>positive control slides for GFP and B-Gal.
>
>We have use AAV contstructs with either GFP or LAC-Z and injected
>into mice.  We have sectioned the tissue and would like to look for
>positive GFP or B-Gal.  It would be nice to have a positive control
>slide.  We plan on visualizing both with and without antibody
>amplification.  I would appreciate any suggestions.
>
>Thank You.
>Rebecca
>------------------------------------
>Rebecca Nishi
>UC Irvine
>Christopher Reeve Foundation SCI Core/Anderson Lab
>1226 GNRF
>Irvine, CA 92697-4540
>rnishi@uci.edu
>Phone/Fax: 949-824-9728
>
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)


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