Re: triple labeling with antibodies

Posted by Stephen Bunnell on
URL: http://confocal-microscopy-list.275.s1.nabble.com/triple-labeling-with-antibodies-tp591231p591247.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We’ve had very good luck doing multicolor (3-4 colors) using CFP with Alexas 488, 568, and 647 on our fixed-dichroic spinning disc. In general, we’ve stuck with one host species for all of our secondary antibodies. That way we can easily pre-block secondary binding sites by supplementing with one whole serum corresponding to the species of origin for your secondaries. We tend to use the highly adsorbed goat-origin secondaries from Invitrogen/Mol Probes. Their isotype specific reagents are very good. We routinely do monoclonal IgG1 with either IgG2a or 1gG2b and a rabbit serum.

I have rarely had the pleasure of finding a primary goat antibody that was clean. Certain Ab suppliers shall not be named publicly...

No commercial interest!

    -Steve



On 12/13/07 2:58 AM, "Shalin Mehta" <[hidden email]> wrote:

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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Stephen C. Bunnell, Ph.D.
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Tufts University Medical School
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