Re: [Histonet] Confirming IHC staining as "real"


I am always surprised how much IHC goes on without these types of 
controls. I think a lot of IHC is meaningless unless you do these types 
of controls whenever such controls are possible.
In the case of an antigen of which we have recombinant form of the 
protein, we compete with the antigen and get clean results.  I think if 
your primary was made against a peptide, competing with soluble peptide 
is always a very convincing control. These "soluble antigen" approaches 
will show specificity.    Another way to have some piece of mind when 
you don't have antigen for competing is and you are using an antibody 
against specific structures or cell types in a tissue is of course to 
make sure other cell types or structures are not staining along. If 
your primary is labeled, the competition with an excess of at least 10 
fold or more unlabeled Ab is a good control as well.  However, 
competing with unlabeled antibody is not going to give you specificity. 
If the labeled antibody is binding to non-specific structures, you 
unlabeled antibody is probably also going to bind to them.

On Mar 3, 2004, at 2:18 PM, Smith, Emily wrote:

> Hi All,
> What techniques are generally used to confirm immunohistochemistry
> staining as real?  If a tissue stains with a labeled primary antibody
> what follow up assays and experiments are done to confirm the staining
> is specific?
> For example, we have tried titering the primary antibody concentration
> to see the staining intensity reduced.  We have tried to "compete" the
> staining away with unlabeled antibody or with the peptide.
> Has anyone tried these or similar approaches?  How were the results?
> Thanks for your help.
> ~Emily
> CuraGen Corporation
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