Sorry for the reply mode, but Patti brings up a good point here.  When
doing a negative control, one should use an isotype matched IgG or whole
IgG (can be purchased from Jackson) at the same concentration in ug/ml as
the antibody.  Some people use normal serum, but that can contain
contaminants.  Using PBS alone in place of an antibody is NOT true negative
control, but a NULL control. NULL controls can test the system to see if
background is coming from the antibody, also in other steps.  

At 09:30 AM 7/5/01 -0700, you wrote:
>If you are using Envision+ for detection, the background can't be from
>biotin. Have you tried titering out your antibodies? I have found Envision
>to be a very clean detection system, and usually no need for even a serum
>block. Take one of your antibodies, and do some doubling titers with it.
>What are you using for a diluent? Also, what did you use in place of the
>primary when you used the spleen without either antibody? How are you
>rinsing in between steps of your procedure? Both of those antibodies can
>usually be used with very little background problems. If I think of anything
>else, I'll send another post.
>
>Patti Loykasek
>Phenopath Laboratories
>Seattle, WA 
>
>
>
>
Gayle Callis
MT,HT,HTL(ASCP)
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
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