triple labeling with antibodies
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Dear all,
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. Thanks Shalin -- ~~~~~~~~~~~~~~~~~~~~~~~~~ Shalin Mehta Graduate Student in Bioengineering, NUS mobile: +65-90694182 blog: shalin.wordpress.com ~~~~~~~~~~~~~~~~~~~~~~~~~~ |
 
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lechristophe |
Re: triple labeling with antibodies
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Dear Shalin, As regards your spectral choice, what is the ratinale behind choosing 514 as the first color ? Is there a reason you don't want to use 488 ? Christophe Leterrier
On Dec 13, 2007 8:58 AM, Shalin Mehta <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, |
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Farid Jalali |
Re: triple labeling with antibodies
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Hello Shalin,
I agree completely with Christophe's' comments. Something that our own lab has started to test for tri-labeling is the Zenon kit/product line from Invitrogen. You can generate your own primary conjugated antibodies quite easily. We often have primary antibodies from the same host that we would like to use together and this product is a very efficient way of doing this without having to search for an antibody from a different host that we would have to test. Labs with stronger ties to chemistry can actually find ways of doing this without this kit. What we have found is that the product most certainly works, but produces a slightly fainter signal. Good Luck Farid On Dec 13, 2007 4:13 AM, Christophe Leterrier <[hidden email]> wrote:
-- Farid Jalali MSc Senior Research Technician/ Lab Manager Dr. Robert Bristow Lab Applied Molecular Oncology Princess Margaret Hospital Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
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Shalin Mehta |
Re: triple labeling with antibodies
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Thanks for comments. We also hope to see GFP with these, so AF488 is not used.
By the way, I actually was also asking a novice explanation of what is involved in high adsorption. What are antibodies adsrobed on (which is the process of accumulating some solute on solid surface that attracts it)? How does it reduce cross-reactivity? Best Shalin On Dec 13, 2007 6:24 PM, Farid Jalali <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Shalin, -- ~~~~~~~~~~~~~~~~~~~~~~~~~ Shalin Mehta Graduate Student in Bioengineering, NUS mobile: +65-90694182 blog: shalin.wordpress.com ~~~~~~~~~~~~~~~~~~~~~~~~~~ |
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In reply to this post by Shalin Mehta
Search the CONFOCAL archive at
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Just a comment on the labeling - are you planning on labeling the
secondaries yourself or are you purchasing labeled antibodies?
Have you tried primary IF? Labeling antibodies with AF is not
difficult, especially if you have a decent amount of IgG (mgs).
Regardless, I'd recommend that, if you don't have a sophisticated
confocal detector together with a great AOTF or an AOBS (say a Zeiss
Meta or Leica SP), you might be better off selecting a lower
excitation wavelength for your 514 antibody. The 514 line on
mixed gas lasers is not very strong and you may have trouble getting
good signal/noise from that probe if you use the 488 line or the 514
line for excitation. Is there any reason why you can't use
AF488? Of course, you may have tried this all out before using
single antibodies and it has not been a problem. Good luck!
I love multiple antibody staining as an approach....
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, -- Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health Oral Infection and Immunity Branch Bldg 30, Room 310 30 Convent Drive Bethesda MD 20892 ph 301-594-0025 fax 301-402-0396 |
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In reply to this post by Shalin Mehta
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No I'm even more skeptical. I'd recommend that, in the
absence of sophisticated excitation control together with good
detection separation (Meta or SP), you try using the GFP and 514
together before making a big commitment in that direction.
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Thanks for comments. We also hope to see GFP with these, so AF488 is not used. On Dec 13, 2007 6:24 PM, Farid Jalali <[hidden email]> wrote:
-- Robert J. Palmer Jr., Ph.D.
Natl Inst Dental Craniofacial Res - Natl Insts Health Oral Infection and Immunity Branch Bldg 30, Room 310 30 Convent Drive Bethesda MD 20892 ph 301-594-0025 fax 301-402-0396 |
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Martin Wessendorf |
Re: triple labeling with antibodies
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In reply to this post by Shalin Mehta
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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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Jeremy Keith Brown |
Re: triple labeling with antibodies
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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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Stephen Bunnell |
Re: triple labeling with antibodies
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In reply to this post by Shalin Mehta
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I have rarely had the pleasure of finding a primary goat antibody that was clean. Certain Ab suppliers shall not be named publicly... No commercial interest! -Steve On 12/13/07 2:58 AM, "Shalin Mehta" <[hidden email]> wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, **************************************************************************** Stephen C. Bunnell, Ph.D. Assistant Professor Tufts University Medical School Department of Pathology Jaharis Bldg., Room 512 150 Harrison Ave. Boston, MA 02111 Phone: (617) 636-2174 Fax: (617) 636-2990 Email: [hidden email] SHIPPING ADDRESS (for packages): Tufts University Receiving 37 Tyler St. Attn: Bunnell/Pathology/Jaharis 524 Boston, MA 02111 |
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Glen MacDonald-2 |
Re: triple labeling with antibodies
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In reply to this post by lechristophe
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Shalin, I second recommendations from Stephan and Christophe. Maybe you are limited to laser laser lines (assume this is a confocal application), but you should try for fluorophores as widely separable as possible to maximize signal with minimal bleedthrough. Avoid Alexa 594, it will co-excite with the 633 and show up in the far red channel. We use AF 488, 568 and 647 or 660 routinely. consider spinning the secondaries for 10 min. at 10,000 rpm in a refrigerated centrifuge then labeling with the supernate to avoid punctate sparklies over you tissue. The Image-iT fx from Molecular Probes greatly reduces AF non-specific tissue binding - this may be particularly important with thicker samples. And, always use negative controls for each of the labels to assess background, autofluorescence and bleedthrough. Regards, Glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] ************************************************************************ ****** The box said "Requires WindowsXP or better", so I bought a Macintosh. ************************************************************************ ****** On Dec 13, 2007, at 1:13 AM, Christophe Leterrier wrote: > Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- > bin/wa?S1=confocal > Dear Shalin, > > I don't know about horse secondary antibodies, but I think you > shouldn't use goat primary and goat secondary on the same sample, > even if they're highly cross-adsorned. We routinely do triple > labeling with goat/mouse/rabbit primary antibodies, using donkey > secondary (donkey anti-goat, anti-mouse and anti-rabbit). > Alternatively, we also use the rat/mouse/rabbit and chicken/mouse/ > rabbit combination with goat secondary antibodies. > > As regards your spectral choice, what is the ratinale behind > choosing 514 as the first color ? Is there a reason you don't want > to use 488 ? > > Christophe Leterrier > > > > On Dec 13, 2007 8:58 AM, Shalin Mehta < [hidden email]> wrote: > Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- > bin/wa?S1=confocal Dear all, > > > 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. > > Thanks > Shalin > > -- > ~~~~~~~~~~~~~~~~~~~~~~~~~ > Shalin Mehta > Graduate Student in Bioengineering, NUS > mobile: +65-90694182 > blog: shalin.wordpress.com > ~~~~~~~~~~~~~~~~~~~~~~~~~~ > |
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Rosemary.White |
Re: triple labeling with antibodies
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In reply to this post by Jeremy Keith Brown
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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 [hidden email] 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" <[hidden email]> 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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Shalin Mehta |
Re: triple labeling with antibodies
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Search the CONFOCAL archive at
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Many thanks to all for comments, recommendations and explanations (especially Martin).
We are going to use Zeiss META system equipped with Argon ion multi-line and green & red HeNe lines. I thought AF514, AF555 and AF633 got very close to available lasers (514nm, 543nm and 632.5nm) and GFP will work with 488nm. Regarding Martin's question about why we want to use alexa dyes - I am under impression that they are one of the brightest and (more importantly for spectral imaging) their spectra are quite pH insensitive. I don't have very stable hands for wet lab, so robust dyes would be good :-). But if others have had better results with other dyes using 488,514,543 and 633nm lines, it will be helpful to consider them. 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. cheers shalin On Dec 14, 2007 4:40 AM, Rosemary White <[hidden email]> wrote: I would also recommend trying fluorescently tagged Fab fragments instead of -- ~~~~~~~~~~~~~~~~~~~~~~~~~ Shalin Mehta Graduate Student in Bioengineering, NUS mobile: +65-90694182 blog: shalin.wordpress.com ~~~~~~~~~~~~~~~~~~~~~~~~~~ |
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Morrison, Ian E |
Re: triple labeling with antibodies
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In reply to this post by Rosemary.White
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Fab fragments are a good idea, but getting them can be very time-consuming and expensive. Some companies offer this service, but the quality is highly variable - naming no names, we've had some total failures to produce labelled Fab that still binds. In our own laboratory, producing Fab from IgG varies enormously; MHC class II works well, CD74 seems to be impossible. You need to make a number of test runs with variable conditions, so shedloads of IgG may be required. If you have to use a 514nm excitation, can I put in a word for Oregon Green 514 (no commercial interest). This can label whole antibody up to ~25probes per IgG with no self-quenching, and on the samples we made using MHC antibodies there was no aggregation and the binding was only slightly compromised. Ian ----------------------------Dr. I.E.G.Morrison {[hidden email]}-------------------------- Dept.Biological Sciences, University of Essex Wivenhoe Park, Colchester, Essex CO4 3SQ ----------------------------Tel: 01206-872246 Fax: 01206-872592-------------------------- |
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xavier Sanjuan |
Re: triple labeling with antibodies
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In reply to this post by Shalin Mehta
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi
Shalin,
tough
I have used AF633 and AF647 with success, Invitrogen people I have talked with
tend to recommend AF647 over AF633 as the fluorophore of choice in the range of
far red.
Xavi.
___________________________________ Xavier
Sanjuan
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Martin Wessendorf |
Re: triple labeling with antibodies
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In reply to this post by Shalin Mehta
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: (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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Jeremy Keith Brown |
Re: triple labeling with antibodies
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal If you do go down the route anti-mouse/anti-rat route you will face cross-reactivity issues. As Martin says, mouse cross-absorbed anti-rat polyclonal (and vice versa) secondaries aren't great, even from Jackson. However, there are some very good mouse anti-rat monoclonals available (try Serotec, BD or SouthernBiotech) which if used in the right order will work for you. Basically you need to label you samples sequentially: mouse and rabbit primaries; then goat anti-mouse and anti-rabbit secondaries; then incubate the samples with 10% normal mouse serum to block any free goat anti-mouse paratopes; then your rat primary (in 10% mouse serum) followed by the mouse anti-rat secondary (in 10% mouse serum). Alternatively, you could indirectly label the sample with the rat and rabbit primaries, block with mouse serum and then come in with the mouse primary labeled with your choice of Alexa flour conjugated Zenon FAB fragments. Obviously, you will need single positive controls for each primary to confirm you're not just detecting spurious cross-reactivity. 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 14 Dec 2007, at 15:18, Martin Wessendorf wrote: > 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: (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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In reply to this post by Martin Wessendorf
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
-- 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 858-822-0461 FAX 858-822-0460 email: [hidden email] |
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Koo, Lily (NIH/NIAID) [E] |
Microscopist Job Posting - NIH
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In reply to this post by Jeremy Keith Brown
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Summary Lockheed Martin Management Systems Designers, Inc., a wholly-owned subsidiary of Lockheed Martin, Inc., is comprised of over 700 professionals who support the diverse technical, scientific, and administrative needs of the Federal Government. Since 1980, we have been providing our public and private sector clients with top-quality information technology (IT), business management, and scientific solutions. Working in partnership with our Civilian Government, national security, and life sciences clients, we deliver innovative, on-target solutions that support our clients' vital missions, goals, and objectives. Among our proven capabilities are systems design, development, and integration; systems engineering; application support, IT infrastructure management; professional services; management consulting; and specialized health and bioinformatics services. We are currently searching for a Microscopist to support the National Institutes of Health (NIH). This opportunity is a permanent, full-time position with Lockheed Martin-MSD and it is on-site at NIH in Bethesda, MD. Major Duties and Responsibilities Assist with the operation of the confocal microscopes as well as perform image analysis and quantification, instrument calibration, data archiving, and evaluation of new software and hardware. Learn new application and software as needed and assist with software/hardware upgrades. Assess user's needs and assist with microscope set-up and operation, image processing, quantitative analysis, and the preparation of figures for publication. Position Requirements § Ph.D. in Biology or other related scientific field. § Familiarity with general light microscopy techniques (such as immunofluorescence, DIC, image capture) and image processing software is required. § Extensive experience with confocal microscopy is required. § Formal training in microscopy and sample preparation is highly preferred. § Excellent verbal and written communication skills. § Ability to juggle multiple projects simultaneously. Please submit CVs and salary requirements to Margaret Hishmeh at [hidden email] |
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