[Histonet] Re: Immunocytochemistry question

From:"Johnson, Teri"

Jerome, you wrote:

>>I'm using ICC to test the phenotype of endothelial cells using
anti-von Willebrand factor.  The primary was produced in rabbit.  To try
and alleviate non-specific binding, as a control I used an isotype IgG
that is also produced in rabbit.  The secondary, FITC, is against
rabbit.  Could using an isotype IgG for blocking from the same species
as the primary lead to excessive non-specific binding??  Would running
the test using IgG from rat possibly reduce non-specific binding since
the secondary is anti-rabbit??<<

I think you're confusing where to use the isotype IgG. That particular
reagent should be used as a negative control on a separate slide, using
the same Ig concentration as your primary antibody (in ug/ml). If you
use this as a serum block prior to antibody incubation (which is what I
think you're asking), your anti-rabbit secondary antibody will recognize
all the sites the rabbit IgG is located and bind everywhere.

You will want to block with a 5-10% solution of normal serum of the
animal your secondary antibody was made in. Your FITC labeled
anti-rabbit antibody was made in what host species? Goat? Donkey? Swine?
Whatever the answer is,  you'll want to use that species' normal serum
as a block prior to incubation in primary antibody. You can also use it
as an antibody diluent for the primary and secondary antibodies if you

Good luck!

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110

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