Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I would also recommend trying fluorescently tagged Fab fragments instead of
full IgGs as secondary labels.
In immunofluorescence on plant tissue, particularly if you are doing
something like immunolabelling of carbohydrates on cereals (as we have been
lately), there is sometimes rather high background with fluorescent IgGs as
secondaries. The explanation I've heard is that the animals in which the
secondaries are raised also produce antibodies to certain components of the
food they eat, which is usually plant material of some sort, often a cereal
grain. Cereals are particularly good at inducing an auto-immune response -
gluten is the classic example.
With Fab secondaries, we get very clean labelling, I guess this would also
be true on animal/human tissues.
cheers,
rosemary
Rosemary White [log in to unmask]
CSIRO Plant Industry ph. 61 (0)2-6246 5475
GPO Box 1600 fax. 61 (0)2-6246 5334
Canberra, ACT 2601
Australia
On 14/12/07 2:48 AM, "Jeremy Keith Brown" <[log in to unmask]> wrote:
> 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
>>
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