Costaining species-specific antibody question - (Feb/20/2012 )

I want to do dual staining on mouse tissues. Antibody 1 is goat polyclonal. Antibody 2 is rabbit polyclonal. The secondary antibody for number 1 is donkey anti-goat 488 and for number 2 is goat anti-rabbit 647. Does the host of the seconday antibodies matter? Will there be non-specific staining with this antibody combination? I typically do mouse/rabbit primary and the secondaries are made in goat so I don't have to worry about it.

Thanks.

Potentially yes there will be cross- reactivity. You should be able to minimise this by doing the rabbit followed by goat secondary before doing the other staining pair. It does double your work, but should give you the result you want.

Be aware that your anti-goat 488 will also stain your goat-anti-rabbit 647.

BioMiha on Tue Feb 21 07:40:10 2012 said:

because of this, i would use the goat:donkey combination before the rabbit:goat.

Thanks! I knew there was something about that combination that needed an extra step!