Re: triple labeling with antibodies

Posted by Martin Wessendorf on
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Shalin Mehta wrote:

> 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.

Dear Shalin--

In triple-labeling, you have 3 primary antibodies, 3 secondary
antibodies, 3 fluorophores, and 3 sets of filters through which you're
viewing the fluorescence.

With regard to the antibodies: your secondary antibody (e.g., horse
anti-goat IgG) is supposed to recognize goat IgG so that it will bind to
your goat primary antibody.  However, suppose it also binds to mouse
IgG.  That would be an example of cross-reactivity by a secondary
antibody.

In principle, the horse anti-goat will bind to ANY goat IgG.  If you
happened to use (as in your example) goat anti-rabbit IgG combined with
the horse anti-goat IgG, the horse anti-goat would bind to the goat
anti-rabbit and you'd observe artifactual colocalization.  So that's a
bad idea.

In general, I'd recommend steering clear of secondary antibodies raised
in goat.  I've seen instances in which goat anti-rabbit IgG recognizes
goat IgG (!) and if such a reagent were used for multiple labeling,
you'd see a lot of artifactual colocalization.

I've had good luck with Jackson ImmunoResearch secondary antibodies.
Bill Stegeman, the owner, has a PhD in biochemistry and they are quite
good about cleaning up their secondary antibodies for use in
multiple-labeling.  --By "cleaning up", I mean doing something to remove
antibodies that might cross-react.  One way to do this would be (for
instance, in the case of a horse anti-rabbit IgG that you didn't want to
cross-react with mouse) to make a column to which mouse IgG was bound
and run your horse anti-rabbit IgG over it.  The "bad stuff" would bind
to the column and the "good stuff" would pass through.  This is probably
what Invitrogen also does to make their "highly absorbed" secondaries.

With regard to direct vs. indirect immunofluorescence--direct labeling
of your primary antibody is possible and isn't that hard, but you'll
want to recharacterize after conjugating it with the fluorophore to be
sure that it still acts the way it did before conjugation.  For that
reason alone, I'd suggest sticking with indirect immunofluorescence
(i.e. immunocytochemistry using secondary antibodies).  In addition,
it's a whole lot more flexible.  --Any particular reason why you want to
use the Alexa dyes?

--I wrote a couple boring and pedantic but fairly complete articles on
multiple labeling many years ago.  If you want to learn more about
characterizing these protocols, they make good bed-time reading:

Wessendorf, M.W., Appel, N.M., Molitor, T.W., and Elde, R.P.:  A method
for the immunofluorescent demonstration of three coexisting
neurotransmitters in rat brain and spinal cord, using the fluorophores
fluorescein, lissamine rhodamine , and 7-amino-4-methylcoumarin-3-acetic
acid.  Journal of Histochemistry and Cytochemistry 38: 1859-1877, 1990.

Wessendorf, M.W.: "Characterization and use of multi-color fluorescence
microscopic techniques."  In: Björklund, A., Hökfelt, T., Wouterlood,
F.G., and van den Pol, A.N. , eds.  Handbook of Chemical Neuroanatomy
Vol. 8: Methods for the analysis of neuronal microcircuits and  synaptic
interactions,  1-45.  Elsevier Science Publishers, Amsterdam, New York,
Oxford, 1990.

There's also this one, in collaboration with Todd Brelje:
T. C. Brelje, M.W. Wessendorf, and R.L. Sorenson: "Multi-color laser
scanning confocal immunofluorescence microscopy: practical application
and limitations."  In: B. Matsumoto, ed.  Methods in Cell Biology,
Volume 38: Cell biological applications of confocal microscopy,  97-181,
Academic Press, New York, 1993.

Good luck!

Martin Wessendorf

--
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