Re: triple labeling with antibodies

Posted by Ella Tour on
URL: http://confocal-microscopy-list.275.s1.nabble.com/triple-labeling-with-antibodies-tp591231p591239.html

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

Dear Confocalists,

Our lab has been getting very good results and no detectable cross-reactivity doing quadruple RNA/DNA/protein stains with the following antibody scheme:

Primary antibodies: mouse, sheep, rabbit, guinea pig

Secondary antibodies: Alexa 488, 555, 594, 647-conjugated anti-mouse, anti-sheep, anti-rabbit and anti-guin. pig, all produced in donkey. Donkey-anti-guinea pig needs to be custom-made (getting donkey anti-rabbit from Jackson, then having Invitrogen put Alexa fluor on it).

This combination was developed by Dave Kosman in our lab, details can be found here: http://superfly.ucsd.edu/%7Edavek/

As mentioned in previous responses, cross-reactivity can often be seen when one works with primaries and secondaries from closely related species. One example that we've repeatedly encountered is the following bad combo: using together donkey anti-sheep and goat-anti-whatever secondary antibodies results in donkey anti-sheep antibodies recognizing  the goat secondaries. On the other hand we've observed no cross-reactivity when detecting mouse and rat primary antibodies, using secondary antibodies (Molecular Probes) which were cross-absorbed against rat and mouse, respectively.

We mount our speciments in Prolong.

I was wondering if anybody has compared Alexa fluorophores against the other commercially available fluorophores, such as Cy3, Cy5, etc in confocal microscopy?

No commercial interest.

Best wishes,

Ella Tour

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

Shalin Mehta wrote:

We were considering purchase goat polyclonal for our antigen, but it seems we will be better off with rat polyclonal and using highly-adsorbed goat secondaries against rabbit,mouse and rat. Zenon kit and Fab fragments also look useful, but will need to read up on them.

There are many different ways of getting satisfactory labeling.  My own recipe for triple-labeling uses Cy2, Red-X (or Cy3, depending on the experiment) and Cy5 secondaries (--I don't use Fab fragments) from Jackson ImmunoResearch that have been cleaned up for minimal cross-reactivity.  (--though you do NOT want to use anti-mouse with minimal cross-reactivity against rat unless you have to use rat as the source for one of your other primaries.  Rat and mouse are immunologically so close that cleaning up against one takes a big bite out of the labeling intensity for the other.)  I'd use the Cy2-labeled secondary in combination with your strongest primary, since the Cy2 signal is a bit weaker than the others.  I would then run the tissue through alcohols and mount in DPX, which gives you a permanent, dry mount.  (I like DPX for those features.)  I've heard others say that they get rapid photobleaching with DPX but I have tissue mounted in it that's been sitting by my microscope for almost 15 years and that I use periodically as test-slides.  If you don't blast it with high illumination power at 60x, the labeling lasts just fine.

Why do you need spectral imaging if you're just looking at dead tissue or cells and the dyes are as well separated as you're describing?  Or is this for when you throw GFP  into the mix?

Good luck!

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455             E-mail: martinw[at]med.umn.edu

-- 

Ella Tour
Department of Cell and Developmental Biology, 0349                    
University of California, San Diego            
9500 Gilman Drive, 4305 Bonner Hall                            
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]