Re: [Histonet] Re: Negative Control Tissue

From:"Andrea Hooper"

I agree with everything said here by Gayle and  I too only accept 
stains which have been run with IgG controls, preferable isotype 
specific IgG controls, at the precise same concentration as the 
primary. In addition, the IgG controls should ideally be from the 
same company as the primary as different companies purify their Abs 
with different methods/buffers etc - this isn't always possible or 
feasible within the budget, but it's a nice thing to aim for.

But I just want to add that another critical negative control besides 
IgG controls for any antibody - especially and importantly when doing 
antibody workups - is to run a tissue processed precisely the same 
way and which is absolutely negative for your antigen alongside your 
test sample. If you see positive staining in your test sample but the 
no-antigen tissue control and IgG controls are negative, well then 
you can be almost 100% sure your stain is real.

Andrea Hooper

>I was taught that a true negative control should not be elimination 
>of the primary antibody (NULL control) but use the immunoglobulin 
>matching the host of the primary antibody and at the same 
>concentration as the primary antibody.  Some people use nonimmune 
>serum, but we prefer using IgG, IgG isotype, or IgM.  I also found 
>out the hard way, in a research project, that using non immune 
>normal serum was unacceptable to reviewers, and the whole, 
>incredibly tedious and difficult immunogold staining had to be 
>repeated before a manuscript was accepted.  A very hard lesson, and 
>now there is a collection of IgG or isotype controls for all 
>immunostaining projects available.
>This immunoglobulin negative control can be used on a patient 
>tissue.  The Ig negative control is there to evaluate nonspecific 
>staining in that particular tissue.  If background occurs, then a 
>source test may be necessary, and with elimination of a primary 
>antibody, that is the first step in a background  source test.
>There is an excellent discussion of negative controls,  tissue or 
>reagent controls,  in the freebie, Dako's Education Guide, 
>Immunohistochenisal staining methods, Fourth Edition and available 
>as pdf on Dako Website. There are several ways of setting up a 
>negative or positive control.  Their comment on page 114 of this 
>manual, verbatim, "Finally, the diluent itself may be used as an 
>alternative, which, however is neither efficient nor desirable" 
>leads me to think the immunoglobulin control is more acceptable.
>Gayle M. Callis
>Bozeman MT 59715

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