RE: [Histonet] negative immuno histochemistry control

From:"Andrea T. Hooper"

I respectfully disagree in most cases. But then again I am a stickler 
for negative controls and precise interpretation of results.

Most of the background one would see with antibodies on tissues 
(assuming the concentrations are reasonable from 0.1-10 ug/ml and 
without ridiculous amplification and one is not using unpurified 
serum but instead somehow purified antibodies or tissue culture 
supernatant) is from Fc binding to endogenous Fc receptors in the 
tissue. Fc is much conserved from animal to animal within the same 
species, so using IgG from another animal is just fine and is in fact 

Antibody diluent is NOT ENOUGH to be robust negative control in your 
interpretation. All that tells you is what background your secondary 
antibody or detection system is contributing. It tells you NOTHING 
about the behaviour of your primary immunoglobulin species whatsoever.

That being said, I am not in a clinical lab I don't know what the CAP 
regulations are specifically so my opinion is merely just that :)

>Although this is a good point, I don't think this is optimal unless the
>normal serum or pre-immune that is used for the negative is from the exact
>same animal/supernatant that the antibody is drawn from with the antibody
>seperated out.  Throwing in something "else" is bringing another factor into
>the equation.  How is a person to know that the "exceptable" negative was
>collected correctly or that it is not contaminated.
>That being said, I myself do the above to remain CAP compliant.  I used to
>do the antibody diluent for whichever antibody I was using for the negative
>control & I still think it's the most logical thing to do.  By using diluent
>as the negative & keeping all other steps/reagents the same as the antibody
>you are running, you are ensuring that it is, indeed, something in the
>concentrated antibody that is causing staining not seen on the negative. 
>Just My Opinion,
>Glen Dawson  BS, HT & QIHC (ASCP)
>IHC Manager
>Milwaukee, WI

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