Re: [Histonet] Negative control for immuno slides

From:Gayle Callis


Removal of the primary is NOT a negative control, you should replace the
primary antibody with the immunoglobulin of host of the primary, ie. if you
work with Rat antiMouse antibody, IgG2a the negative control is either Rat
IgG which contains IgG2a isotype or use Rat IgG 2a and at the same
concentration as the primary antibody. You must have primary host IgG in
the system in order to know IF this immunoglobulin is crossreacting to
tissue being stained. The negative control section must be treated exactly
as you treat the unknown tissue, including all steps of your IHC protocol. 

OR you can use an irrelevant antibody of same IgG isotype that does not
recognize you antigen OR you can use a transgenic knockout animal known to
NOT express the antigen you are interested in staining. However, on the
latter, knockout animal, we have found the host IgG sometimes does cross
react, so we still do the isotype matched control or immunoglobulin
control.  We do NOT use normal serums as they can contain nonspecific
antibodies - presenting background problems.   Jackson Immunoresearch has a
wonderful array of different species immunoglobulins that are very
inexpensive and clean. 

If you have several antibodies with same host of primary, and at same
antibody concentrations, you can get away with one control for many
antibodies.  DAKO manual by Boenisch discussion on this is very clear, and
a good read, go to DAKO website, click on support and read the pdf file on

Removal of primary is considered a NULL control, and has its use when you
want to do a reagents test for background. 


At 09:22 PM 5/6/2004 -0700, you wrote:
>We do our negative reagent control by omitting the primary antibody. The 
>number of negative
>slides we run depends on the pre-treatment of the antibodies requested on 
>the case. We run one
>negative slide for each method i.e. no pre-treatment, protease and pressure 
>cook. Are we doing
>more negative control slides than what is required ? I know some people just 
>do one negative
>slide per case. Any comments on the proper way of running negative immuno 
>controls? Thanks
>in advance for your inputs !
>Anissa Choi
>Burnaby Hospital
>Burnaby, B.C.
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