We do this routinely. The keys will be

1) Good primary antibodies- preferably raised in three different species. If you have good primary antibodies from different species and have them titrated (diluted) properly, you can apply all three simultaneously rather than sequentially. This will save you a lot of time.

1A)  A potential danger to watch out for in CNS tissues is that long exposure to detergents like Triton-X100 can lead to increased autofluorescent background, especially in CNS areas that have high myelin content. This autofluorescence is is worst in the green channel. If you can apply your primary antibodies simultaneously, this will help to minimize this problem.

2) Highly specific secondary antibodies that recognize ONLY their target species. This cannot be stressed enough. Any cross-reactivity will lead to false-positive results.  It is worth the money to buy secondary antibodies that have been highly cross-adsorbed to remove Igs that cross-react with other off-target species. Check the the catalog information and be sure that you are buying the right antibody-- do not assume that any old secondary antibody is sufficiently specific. We have had good luck with highly cross-adsorbed antibodies from Jackson ImmunoResearch and Molecular Probes in this regard. Again, if you have good secondary antibodies and have them titrated (diluted) properly, you can apply all three simultaneously rather than sequentially. This will save you a lot of time.

3)  Good matching of your fluorochromes to the fluorescent filters in your epifluorescence system (or the illumination system in your confocal microscope if you are doing confocal work). The Molecular Probes website offers a great tool for checking these types of things out using their "SpectraViewer" applet, but you'll need to know the info for your microscope's filter/illumination system.

   For our microscopes we use: DAPI, AlexaFluor 488, AlexaFluor 568, and AlexaFluor647 routinely.   Other microscopes and illumination systems might be better tuned for different fluorophore combinations.

4)  If you are imaging by confocal microscopy, I would encourage you to image each channel independently (called "sequential scan" in the Olympus Fluoview software) rather than simulatneously to prevent spectral bleedthrough between fluorescence channels.

Hope this helps. Good luck,

Dave Sherry

Dear Marina,

I haven't done 4 color, only two color plus Hoechst for DNA.  However, the principal for multicolor labeling is the same; it will just be more involved in your case.

For multicolor immunofluorescence, you should plan to use secondary antibodies that are made in the same animal in order to recognize the different primary antibodies.  That is, if you have mouse, rabbit, and chicken primary antibodies, your anti-mouse, anti-rabbit, and anti-chicken should all be raised in the same animal (many times it's goat or donkey, but others are available, too).  It is also helpful to high quality secondary antibodies that have minimal cross-reactivity to other the other species Ig.  That is, for my example above, the donkey anti-mouse should have minimal cross reactivity to rabbit and chicken.  Once you have that worked out, the easiest blocking protein to use is normal serum of the animal in which the secondary antibodies are raised (donkey for my example).  Sometimes BSA will work too, but it depends on the quality of the BSA.

One resource that I make use of is the information in the Jackson ImmunoResearch website:

https://www.jacksonimmuno.com/technical/products

There are also considerations for compatibility of labeling parameters:  for fixation (4 % paraformaldehyde good for all antigens; is antigen retreival necessary?), tissue permeabilization  (Triton X-100, methanol, saponin, or something else?),  which color fluorochrome to use for which probe (should the least abundant use the brightest fluorochrome?).  It's helpful to work out conditions for each individual probe in your hands first, and then try to combine them.  

You will also need to pick fluorochromes with excitation and emission spectra that have minimal overlap, and an instrument that can discriminate between each fluorochrome.  One tool that's helpful for designing this is Omega Filter's Curvomatic:

http://www.omegafilters.com/curvomatic/

Good Luck!

Jill

Dear Marina,

We did four-color immunofluorescence, check this:

http://link.springer.com/article/10.1007%2Fs00429-016-1217-x

Good luck,

Kata

I have done 3 color + DAPI. As Jill said, primary antibodies (PAb) should from different animal e.g rabbit anti mouse, rat anti mouse, chicken anti mouse and the secondary antibodies (SAb) should be made in the same animal e.g donkey anti rabbit Cy2, donkey anti rat Cy3, donkey anti chicken Cy5.

For blocking you can use normal donkey Serum 5 % (Serum of animal, which secondary antibodies made in). PAb you can incubate at the same time all three AB, the SAb also.

If you have PAb from the same animal, you can also use to stain 2 or 3 color, but you have to use FAb secondary antibody but not IgG or F(AB)2 antibodies. And staining protocol is longer, you should incubate each couple antibodies in sequence. e.g (rabbit anti mouse,  then donkey anti rabbit Cy2 ) ( rat anti mouse  and then donkey anti rat Cy3).......

Good Luck

DL

We do this routinely. The keys will be

1) Good primary antibodies- preferably raised in three different species. If you have good primary antibodies from different species and have them titrated (diluted) properly, you can apply all three simultaneously rather than sequentially. This will save you a lot of time.

1A)  A potential danger to watch out for in CNS tissues is that long exposure to detergents like Triton-X100 can lead to increased autofluorescent background, especially in CNS areas that have high myelin content. This autofluorescence is is worst in the green channel. If you can apply your primary antibodies simultaneously, this will help to minimize this problem.

2) Highly specific secondary antibodies that recognize ONLY their target species. This cannot be stressed enough. Any cross-reactivity will lead to false-positive results.  It is worth the money to buy secondary antibodies that have been highly cross-adsorbed to remove Igs that cross-react with other off-target species. Check the the catalog information and be sure that you are buying the right antibody-- do not assume that any old secondary antibody is sufficiently specific. We have had good luck with highly cross-adsorbed antibodies from Jackson ImmunoResearch and Molecular Probes in this regard. Again, if you have good secondary antibodies and have them titrated (diluted) properly, you can apply all three simultaneously rather than sequentially. This will save you a lot of time.

3)  Good matching of your fluorochromes to the fluorescent filters in your epifluorescence system (or the illumination system in your confocal microscope if you are doing confocal work). The Molecular Probes website offers a great tool for checking these types of things out using their "SpectraViewer" applet, but you'll need to know the info for your microscope's filter/illumination system.

   For our microscopes we use: DAPI, AlexaFluor 488, AlexaFluor 568, and AlexaFluor647 routinely.   Other microscopes and illumination systems might be better tuned for different fluorophore combinations.

4)  If you are imaging by confocal microscopy, I would encourage you to image each channel independently (called "sequential scan" in the Olympus Fluoview software) rather than simulatneously to prevent spectral bleedthrough between fluorescence channels.

Hope this helps. Good luck,

Dave Sherry

Dear Marina,

I just got a notice for a BioTechniques webinar on multi-color imaging.  If you need more information than what we have been able to provide in our answers, you might want to check it out.

https://engage.vevent.com/index.jsp?eid=7110&seid=15&code=banner

Cheers!

Jill