Re: What I'm using Nonspecific binding to mucus
|From:||Phil Bergin <email@example.com>|
Forgot to say what I was using. I tend to get the problem whatever antibodies
I use, but only occasionally, I suppose it depends on the amount of mucus in
the biopy. But here are the details:
At the moment I'm using human tissue, but we tend to get the same problem with
our mouse tissue as well.
I start by blocking with glucose oxidase (for endogenous peroxidase), then use
mouse anti-human Ab's diluted in PBS with 1% goat serum. I add a biotinylated
goat anti-mouse Ab diluted in PBS with 5% human AB serum, then the standard
avidin-biotin kit. Detection with AEC substrate.
We also use the same system with DAB as the substrate and get the non-specific
binding as well,
Thanks for the help,
Lynn Gardner wrote:
> What kind of blocking steps are you already doing prior to your primary
> antibody and what are you using to dilute your primary. What are you using
> for your secondary and what are you using to dilute it. Also I assume this
> is human tissue and you are using a (mouse, rabbit, goat) anti-human
> antibody for your primary?
> If you let me know this I can help you with the background issues.
> Lynn Gardner
> At 10:01 AM 6/7/01 +0200, you wrote:
> >Thanks to everyone who gave me advice on brittle blocks. Now I have
> >another question. I stain gastric tissue looking for macrophages and
> >matrix metalloproteinases, unfortunately with some of the antibodies I
> >get a lot of non specific binding to mucus withine the tissue, does
> >anyone know a good way of blocking binding to gastric mucus?
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