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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear  Shalin,
I second recommendations from Stephan and Christophe.  Maybe you are  
limited to laser laser lines (assume this is a confocal application),  
but you should try for fluorophores as widely separable as possible  
to maximize signal with minimal bleedthrough.  Avoid Alexa 594, it  
will co-excite with the 633 and show up in the far red channel.  We  
use AF 488, 568 and 647 or 660 routinely.  consider spinning the  
secondaries for 10 min. at 10,000 rpm in a refrigerated centrifuge  
then labeling with the supernate to avoid punctate sparklies over you  
tissue.  The Image-iT fx  from Molecular Probes greatly reduces AF  
non-specific tissue binding - this may be particularly important with  
thicker samples.  And, always use negative controls for each of the  
labels to assess background, autofluorescence and bleedthrough.

Regards,
Glen



Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
(206) 616-4156
[log in to unmask]

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On Dec 13, 2007, at 1:13 AM, Christophe Leterrier wrote:

> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- 
> bin/wa?S1=confocal
> Dear Shalin,
>
> I don't know about horse secondary antibodies, but I think you  
> shouldn't use goat primary and goat secondary on the same sample,  
> even if they're highly cross-adsorned. We routinely do triple  
> labeling with goat/mouse/rabbit primary antibodies, using donkey  
> secondary (donkey anti-goat, anti-mouse and anti-rabbit).  
> Alternatively, we also use the rat/mouse/rabbit and chicken/mouse/ 
> rabbit combination with goat secondary antibodies.
>
> As regards your spectral choice, what is the ratinale behind  
> choosing 514 as the first color ? Is there a reason you don't want  
> to use 488 ?
>
> Christophe Leterrier
>
>
>
> On Dec 13, 2007 8:58 AM, Shalin Mehta < [log in to unmask]> 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
>
> -- 
> ~~~~~~~~~~~~~~~~~~~~~~~~~
> Shalin Mehta
> Graduate Student in Bioengineering, NUS
> mobile: +65-90694182
> blog: shalin.wordpress.com
> ~~~~~~~~~~~~~~~~~~~~~~~~~~
>