Re: [Histonet] Negative control IHC

From:Gayle Callis


Reagent negative or buffer is NOT a true negative control.  You need to 
have the immunoglobulin, at the same working concentration as your primary 
antibody, which  matches a primary antibody host immunogloblin (either 
whole or an immunoglobulin isotype).  The only time we use PBS, then called 
a NULL control, is when we test our staining system for background 
sources.  IF you use PBS as a negative control in a submitted manuscript, a 
reviewer could very well say a proper negative was not used and you would 
have to repeat the work.  This happened to us when using normal serum, when 
the reviewer demanded an immunoglobulin negative control, in particular, an 
immunoglobulin isotype.  This resulted in our no longer using normal serums 

Some people in the clinical area use an irrelevant antibody that will not 
stain anything in the tissue but provides the immunoglobulin needed. 
However, with murine or rat species, it is very difficult to find an 
irrelevant rat or hamster antimouse that doesn't light up some mouse/rat 
tissue component.  Consequently, we use an immunoglobulin control from Rat 
or hamster (paying attention to WHICH hamster species here) immunoglobulin, 
IgG, IgG isotype, IgM, Armenian Hamster Ig, Goldern Syrian IgG.  Many of 
these are conjugated to biotin and/or fluorophores for special applications 
when working with a biotinylated primary or a fluorophore that is directly 
conjugated to the primary.

You can purchase negative immunglobulin controls readily from any vendor 
selling antibodies.

You will find more answers to your question in Histonet archives, as this 
has been asked many times.

At 12:07 AM 6/18/2007, you wrote:
>Morning Histonetters
>What do you use for negative controls for FFPE tissue in IHC, apart from 
>the reagent negative.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610

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