1 reply to this topic

[Rnotk]

Hi, I have a little problem with IHC.
The problem is, as I said on title, non-specific binding.

I am staining mouse tissue samples (frozen section), but due to the antibody availability, I have to use some antibodies which is made in mouse,
so the secondary antibody will be anti-mouse and this is creating huge background under microscope.

I tried some blocking but no luck so far.
So does anyone know good blocking that I can try?

Also, some people suggested me that I might be able to use goat anti-mouse IgG fragment to cover the mouse IgG before secondary,
but could not find protocol for that, so if you know protocol and possible let me know, that really help me too.

Thanks in advance.

[klinmed]

Rnotk, on Aug 12 2009, 05:18 AM, said:

Hi, I have a little problem with IHC.
The problem is, as I said on title, non-specific binding.

I am staining mouse tissue samples (frozen section), but due to the antibody availability, I have to use some antibodies which is made in mouse,
so the secondary antibody will be anti-mouse and this is creating huge background under microscope.

I tried some blocking but no luck so far.
So does anyone know good blocking that I can try?

Also, some people suggested me that I might be able to use goat anti-mouse IgG fragment to cover the mouse IgG before secondary,
but could not find protocol for that, so if you know protocol and possible let me know, that really help me too.

Thanks in advance.

There are many protocols and also commercial kits/reagents to sort out this problem.  Google "IHC mouse on mouse" and you will find lots of possibilities.

Hope this helps.