RE: Urgent question
|From:||"Bennett, Catherine (Katie)" <firstname.lastname@example.org>|
Have you tried decreasing your primary concentration and/or switching to an
overnight incubation at 4C?
It may be non-specific secondary binding, though. Are you making up your
secondary in the blocking solution?
Also, you might want to look into buying a secondary antibody that is mouse
adsorbed. Or you can make it adsorbed yourself by adding 20ul/ml normal
mouse serum to the secondary antibody solution. Let this solution sit at 4C
overnight before using. This will hopefully bind up any secondary
antibodies that might non-specifically bind to mouse tissues. You may find
however that you might need to increase your secondary concentration since
much of it may get bound up in the adsorption process.
I'd recommend the Vector Lab's website which has an awesome troubleshooting
guide for helping determine where non-specific background staining might be
coming from. See www.vectorlabs.com and hit the Immunohistochemestry link
to find the troubleshooting guide.
Catherine "Katie" Bresee Bennett
Sr. Research Technologist
Lovelace Respiratory Research Institute
Albuquerque, New Mexico
> -----Original Message-----
> From: Lynn Gardner [SMTP:email@example.com]
> Sent: Thursday, January 25, 2001 6:17 PM
> To: Histonet@pathology.swmed.edu
> Subject: Urgent question
> Dear Friends,
> We are staining for anitbodies in nuede mice. They have been injected with
> rat cells and we want to demonstrate the antibody in the rat cells. The
> antibody is a rabbit anti-rat antibody. The problem is we get staining in
> the tissue but everything is staining. The more worrysome problem is that
> the Rabbit IgG negative control is also staining vibrantly positive even
> more so than the antibody stained tissue. Does this mean that the rabbit
> anti-rat antibody and the Rabbit IgG are cross reacting with the mouse
> tissue? or something else?
> I block for Hydrogen peroxide, avidin and biotin and use a protein block
> that is serum free. I can't figure this one out. Any hints would be most
> Thanks gang,
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