Barb,
i know this sounds crazy but i used to make my own secondary reagent and we always
added 40% human serum and it worked really well with no background.
Patsy

Barbara Wright wrote:

> 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