Marilyn,
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
anymore.
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.
>Thanks
>Marilyn
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
|