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Costaining species-specific antibody question

costaining species antibody IF IHC

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4 replies to this topic

#1 PaigeD

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Posted 20 February 2012 - 12:59 PM

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.

#2 bob1

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Posted 20 February 2012 - 03:11 PM

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.

#3 BioMiha

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Posted 20 February 2012 - 11:40 PM

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

#4 mdfenko

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Posted 21 February 2012 - 12:39 PM

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

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

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Posted 24 February 2012 - 12:45 PM

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





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