triple labeling with antibodies

Posted by Shalin Mehta on
URL: http://confocal-microscopy-list.275.s1.nabble.com/triple-labeling-with-antibodies-tp591231.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all,


I am getting out of my depth about triple labeling with antibodies. We have three molecules that we want to label and see simultaneously with AF514, AF555 and AF633. We have decided to use goat, mouse and rabbit antibodies for three antigens. Now, it seems pretty straightforward to use e.g. horse anti-goat, horse anti-mouse and horse anti-rabbit secondary antibodies (I didn't know about the issue of cross reactivity.)  I noticed on Invitrogen website that they offer 'highly adsorbed' secondary antibodies (goat anti-mouse and goat anti-rabbit) for multiple labeling experiments. Does it mean that we should be safe using, e.g., horse anti-goat, highly adsorbed goat anti-rabbit and highly adsorbed goat anti-mouse? I am not sure what cross reactivity means and what logic dictates choice of secondary antibodies.
Any explanation will be helpful.

Thanks
Shalin

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Shalin Mehta
Graduate Student in Bioengineering, NUS
mobile: [phone]
blog: shalin.wordpress.com
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