http://confocal-microscopy-list.275.s1.nabble.com/triple-labeling-with-antibodies-tp591231p591234.html
secondaries. The explanation I've heard is that the animals in which the
grain. Cereals are particularly good at inducing an auto-immune response -
CSIRO Plant Industry ph. 61 (0)2-6246 5475
GPO Box 1600 fax. 61 (0)2-6246 5334
> Search the CONFOCAL archive at
>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>
> We use FAB fragment - Primary antibody complexes if we need to perform
> complex multiplex-labeling.
>
> On their own...
> Brown, J. K., A. D. Pemberton, S. H. Wright, and H. R. Miller. 2004.
> Primary antibody-Fab fragment complexes: a flexible alternative to
> traditional direct and indirect immunolabeling techniques. J Histochem
> Cytochem 52:1219-1230.
>
> or in combination with standard indirect ICC......
> Brown, J. K., P. A. Knight, A. D. Pemberton, S. H. Wright, J. A. Pate,
> E. M. Thornton, and H. R. Miller. 2004. Expression of integrin-alphaE
> by mucosal mast cells in the intestinal epithelium and its absence in
> nematode-infected mice lacking the transforming growth factor-beta1-
> activating integrin alphavbeta6. The American journal of pathology
> 165:95-106.
>
> We use usually use Jackson Immuno FAB fragments, but you can get Alexa
> Fluor labelled FAB fragments from Invitrogen under the Zenon brand
> name. That said, assuming you are not compromising your choice of
> primaries by using mouse, rabbit and goat antibodies, invitrogen's
> donkey Alexa Fluor conjugates will work well in the context you
> describe and there should be minimal cross-reactivity between these
> three species. However, if you have the choice, go for the highly
> absorbed versions.
>
> Cheers,
> Jeremy
>
> Dr Jeremy K Brown
> The University of Edinburgh
> Easter Bush Veterinary Centre
> Easter Bush
> Midlothian
> EH25 9RG
>
> Tel: ++44 131 650 7348
>
>
>
>
> On 13 Dec 2007, at 15:01, Martin Wessendorf wrote:
>
>> Search the CONFOCAL archive at
>>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>>
>> 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: (612) 626-0145
>> Assoc Prof, Dept Neuroscience lab: (612) 624-2991
>> University of Minnesota Preferred FAX: (612) 624-8118
>> 6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
>> Minneapolis, MN 55455 E-mail: martinw[at]med.umn.edu
>>