I think putting human serum on the sections to "block" would INCREASE the
background considerably.  The second antibody (anti-human) would bind the
human Ig's added to the sections.

Ralph

-----Original Message-----
From: Barbara Wright [mailto:bwright@gene.COM]
Sent: Monday, December 17, 2001 3:00 PM
To: rueggp
Cc: Crochiere, Steve; Histonet (E-mail)
Subject: Re: human-human ihc


Patsy;

If Steve puts 40% human serum in his secondary reagent which, if I am
understanding
correctly should be a anti-human IgG - the 40% human serum should bind to
all of
the anti-human IgG in the secondary reagent leaving no binding for the
primary
antibody.  Steve can put the 40% human serum in his protein blocking reagent
and in
his primary antibody - but I would leave it out of the secondary.  Am I
reading it
correctly?

Thanks
Barb Wright

rueggp wrote:

> what i would do for this is use lots of normal human serum to block with
before
> the primary antibody and again before the secondary, i would also put
human
> serum in my secondary reagent.  by a lot i mean about 40% solution.
> patsy ruegg
>
> "Crochiere, Steve" wrote:
>
> > Hi,
> > Does anyone out there have an IHC procedure for staining human tissue
with
> > human Mab's? I have tried to do this by blocking endogrnous peroxidase
with
> > H2O2,  IgG activity with a FAB fragment, protein with normal serum, and
> > biotin with the vectorlabs biotin blocking sysytem, and still have so
much
> > background and non-specific staining in the negative controls (IgG whole
> > molecule) that the slides are unreadable.
> > Any suggestions would be greatly appreciated.
> > Thanx,
> > steve