NEW! 4-Color Imaging with DyLight 405 Conjugates

Our new DyLight 405-conjugated secondary antibodies are excited maximally at about 400 nm and fluoresce with a peak at about 421 nm (Figure 1). They are very bright and photostable when used with confocal microscopes equipped a 405 nm laser and appropriate emission filters (Figure 2).

Figure 1. Excitation (violet) and emission (blue) spectra for DyLight 405 conjugated to an affinity-purified secondary antibody from Jackson ImmunoResearch. Quantitative comparisons should not be made since peak heights have been normalized. Spectra were obtained with an M-Series spectrofluorometer system from Photon Technology International, Inc.

Figure 2. Mouse retinal tissue stained with DyLight 405 (pseudo-colored green)-donkey anti-rabbit IgG (JIR) with rabbit anti-GFAP (DAKO), DyLight 488 (pseudo-colored red)-donkey anti-mouse IgG (JIR) with mouse anti-neurofilament (Abcam), Cy3 (pseudo-colored blue)-donkey anti-chicken IgG (JIR) with chicken anti-vimentin (Chemicon), and DyLight 649 (pseudo-colored white)-donkey anti-goat IgG (JIR) with goat anti-collagen IV (Chemicon). Sections were mounted in n-propyl gallate-glycerol for imaging with an Olympus Flouview 1000 Laser Scanning confocal microscope equipped with a 405 nm laser and a krypton/argon laser. Images are courtesy of Gabe Luna, Neuroscience Research Institute, UC Santa Barbara.

Under these conditions, it is possible to perform effective 4-color imaging with good color separation, good photostability, and high sensitivity in both aqueous and permanent mounting media. The combination of DyLight 405, DyLight 488, Rhodamine Red-X, and DyLight 649 provides for maximum color separation (Figure 3).

Figure 3. Emission spectra of DyLight 405 (blue), DyLight 488 (green), Rhodamine Red-X (red), and DyLight 649 (brown). This figure illustrates the relative shape and position of each fluorophore emission peak following conjugation to antibodies. It shows that effective 4-color imaging can be performed with maximum color separation using these dyes. Quantitative comparisons should not be made since peak heights have been normalized. All spectra were obtained with an M-Series spectrofluorometer system from Photon Technology International, Inc.

Other 4-color dye combinations, which may be equally effective but have slightly less color separation, include DyLight 405, DyLight 488, DyLight 549 (or Cy3), and DyLight 649 (Figure 4).

Figure 4. Mouse retinal tissue stained with DyLight 405 (pseudo-colored green)-donkey anti-rabbit IgG (JIR) with rabbit anti-GFAP (DAKO), DyLight 488 (pseudo-colored red)-donkey anti-mouse IgG (JIR) with mouse anti-neurofilament (Abcam), Cy3 (pseudo-colored blue)-donkey anti-chicken IgG (JIR) with chicken anti-vimentin (Chemicon), and DyLight 649 (pseudo-colored white)- donkey anti-goat IgG (JIR) with goat anti-collagen IV (Chemicon). Sections were mounted in n-propyl gallate-glycerol for imaging with an Olympus Flouview 1000 Laser Scanning confocal microscope equipped with a 405 nm laser and a krypton/argon laser. Images are courtesy of Gabe Luna, Neuroscience Research Institute, UC Santa Barbara.

The following table lists highly adsorbed affinity-purified secondary antibodies, streptavidin, and immunoglobulin controls conjugated with DyLight 405 as offered by Jackson ImmunoResearch for use only in 4-color labeling protocols, using a 405 nm laser-equipped confocal microscope. For a complete listing of other DyLight 405 conjugates for 1-, 2-, and 3-color labeling, see other tables in this Web site.

PerCP Conjugates for Flow Cytometry

PerCP is a fluorescent peridinin-chlorophyll protein complex isolated from dinoflagellates. We offer the form found in Dinophyceae sp. with a molecular weight of about 35.5 kDa. It has a broad spectrum of excitation with a main peak at 472 nm, and a long Stokes shift to an emission peak at 677 nm (Figure 1).
Figure 1. Relative shape and position of spectra in the peak region of excitation (blue) and emission (purple) for PerCP conjugated to an affinity-purified secondary antibody from Jackson ImmunoResearch. Quantitative comparisons should not be made since peak heights have been normalized. Spectra were obtained with an M-Series spectrofluorometer system from Photon Technology International, Inc.

PerCP conjugates of highly adsorbed secondary antibodies are offered to label unconjugated primary antibodies, and PerCP-streptavidin is offered to label biotinylated primary or secondary  antibodies  (Figures 2 and 3).  Two practical  labeling  protocols are
Figure 2. Human peripheral lymphocytes were stained either directly with PerCP-conjugated anti-CD3 (orange) from BioLegend, or indirectly with biotin-conjugated anti-CD3 (B-D Pharmingen) and PerCP-streptavidin either from Jackson ImmunoResearch (blue), BioLegend (purple), or B-D Pharmingen (red). For further comparison, cells were stained with unconjugated anti-CD3 (B-D Pharmingen) and PerCP-conjugated secondary antibody from Jackson Immuno-Research (green). Stained cells were analyzed in a dual-laser FACSCalibur flow cytometer (Becton Dickinson).		Figure 3. Human peripheral lymphocytes were stained with unconjugated anti-CD3 (B-D Pharmingen) and PerCP-conju-gated secondary antibody from Jackson ImmunoResearch (green), or with biotin-conjugated anti-CD3 (B-D Pharmingen) and PerCP-streptavidin from Jackson ImmunoResearch (blue). For further comparison, cells were stained with unconjugated anti-CD3 (B-D Pharmingen), biotinylated secondary antibody and PerCP-streptavidin from Jackson ImmunoResearch (red). Stained cells were analyzed in a dual-laser FACSCalibur flow cytometer (Becton Dickinson)

possible with these products. Compared with a single-step PerCP-conjugated primary antibody (Figure 2), about the same level of fluorescence is obtained with a two-step procedure using a biotinylated primary antibody and PerCP-conjugated streptavidin. A consistent, slightly higher signal is achieved by using an unconjugated primary antibody and PerCP-conjugated secondary antibody. Although three-step procedures are usually undesirable for flow cytometry, a somewhat greater amplification may be obtained with unconjugated primary antibody, biotinylated secondary antibody, and PerCP-conjugated streptavidin (Figure 3).

Figure 4. Excitation spectra (left) for PerCP-(blue), DyLight 488/FITC- (green), R-PE- (red), and APC- (brown) conjugated secondary antibodies from Jackson ImmunoResearch. Emission spectra (right) for DyLight 488/FITC- (green), R-PE- (pink), APC- (brown), and PerCP- (purple) conjugated secondary antibodies from Jackson ImmunoResearch. Quantitative comparisons should not be made since peak heights have been normalized. All spectra were obtained with an M-Series spectrofluorometer system from Photon Technology International, Inc.

PerCP conjugates may be used alone or with DyLight 488 (or FITC) and R-PE for one- to three-color analyses with a single-laser flow cytometer equipped with an argon laser emiting at 488 nm. Up to four-color analyses with low compensation are easily achieved by adding APC-conjugated antibodies with 633 or 635 nm excitation provided by a dual-laser flow cytometer (Figure 4).

See Table of NEW PerCP Conjugates here.