Re: triple labeling with antibodies

Posted by Martin Wessendorf on
URL: http://confocal-microscopy-list.275.s1.nabble.com/triple-labeling-with-antibodies-tp591231p591236.html

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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
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