Re: Urgent question

From:"C.M. vander Loos" <>

>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,

Although I am not really experienced with this type of tissue, I do know
that mouse and rat immunoglobulins are very similar because of their close
genetical relationship. As a result an anti-rat Ig antibody will cross
react with mouse Ig for at least 50% (and the other way around). In your
case it seems to me that the rabbit anti-rat antibody is reacting with
mouse Ig in the tissue section.
In order to prevent this you may try to mix the rabbit anti-rat antibody
with 10-20% normal mouse serum, at least 30 min (at room temperature)
before actual application to the tissue section. All rabbit anti-rat
antibodies that tend to react with mouse Ig will be blocked then.

Good luck,

Chris van der Loos
Amsterdam, The Netherlands

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