RE: Negative Controls Immunos

From:Gayle Callis <>

Using buffer in place of antibody is NOT a negative control (called a
"null" control) and used to test for nonspecific staining by a antibody.
You must use host immunoglobulin isotype matched control, normal serum, or
host of antibody IgG such as a rat anti mouse CD4, we would use rat IgG
isotype matched control (IgG2a) or rat IgG. An irrelevant antibody can also
be used.  It must be remembered that pooled normal serums may contain
antibodies and could cause nonspecific staining at times although have been
used.  Isotype matched controls are preferred, and if you are publishing,
it is best to use these or end up repeating the work, been there, done
that, what a chore! 

Vendors do sell negative controls (host immunoglobulins, etc) ready to use.
 Vector is example.   

There is a systems test for nonspecific background staining where PBS is
substituted systematically for each reagent, very nice for solving
nonspecific background staining at every step in your protocol.

At 01:31 PM 2/1/01 -0500, you wrote:
>Is anyone still using buffer and not serum for negative controls? Thanks, j
>Joyce Weems
>Pathology Manager
>Saint Joseph's Hospital of Atlanta
>	-----Original Message-----
>	From: []
>	Sent:	Wednesday, January 31, 2001 10:15 PM
>	To:;
>	Subject:	Re: Negative Controls Immunos
>	Hi, Donna,  we do not run a negative with each antibody.  It would
>run the 
>	cost of reagents and tech time up too high.  We do run a positive
>with each 
>	antibody (our control is on the same slide as the unknown) .  For a
>	I like to use a sausage various types of tissue... prostate, small
>	appendix, tonsil, placenta,  etc.    and we use a Rabbit normal
>serum or a 
>	Mouse normal serum depending on what antibodies are run.  And we
>only run one 
>	negative.  Or if the antibodies for that case involve rabbit and
>mouse, we 
>	run one mouse normal serum and one rabbit normal serum.     We just
>had a CAP 
>	inspection and our inspectors had no problems with the way we did
>	Pearl Gervais
Gayle Callis
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)

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