Re: triple labeling with antibodies

Posted by rjpalmer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/triple-labeling-with-antibodies-tp591231p591248.html

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Just a comment on the labeling - are you planning on labeling the secondaries yourself or are you purchasing labeled antibodies?  Have you tried primary IF?  Labeling antibodies with AF is not difficult, especially if you have a decent amount of IgG (mgs).  Regardless, I'd recommend that, if you don't have a sophisticated confocal detector together with a great AOTF or an AOBS (say a Zeiss Meta or Leica SP), you might be better off selecting a lower excitation wavelength for your 514 antibody.  The 514 line on mixed gas lasers is not very strong and you may have trouble getting good signal/noise from that probe if you use the 488 line or the 514 line for excitation.  Is there any reason why you can't use AF488?  Of course, you may have tried this all out before using single antibodies and it has not been a problem.  Good luck!  I love multiple antibody staining as an approach....

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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Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health
Oral Infection and Immunity Branch
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