Jackson Immuno Research Inc.
specializing in secondary antibodies and conjugates

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Multiple Labeling

With BV421™ & BV480™

  • Combine with AF488, RR-X, and AF647 for 5-color imaging
  • Compatible with commonly used filter sets
  • Switch nuclear stain from DAPI to DRAQ5 for additional labeling options

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Whole IgG Affinity-Purified Secondary Antibodies

"We only use secondary antibodies from Jackson ImmunoResearch whenever possible! Customer service is also excellent and the price point for their products is very competitive."

Elizabeth Soberg, University of Washington

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Sequential Labeling

Importance of Sequential Labeling
and Titrating Primary vs. Target Antigen

Figures 1-5 show how an optimized multiple labeling method can improve results. The potential for cross-reactive interactions of the FabuLights™ is minimized by sequentially labeling the less abundant target antigen first, and by titrating Fab-labeled complexes vs. target antigen.

Blood was collected from normal human donors and treated with lysis buffer. Cells were centrifuged, washed, and resuspended in isotonic PBS + 0.5% BSA.

Complexes were formed using Fab anti-Fc:primary at 3:1 ratio (equal weight ratios). Alexa Fluor® 488-conjugated Fab Goat Anti-Mouse IgG1, Fcγ fragment specific was complexed with Mouse Anti-Human CD3 (BD #555330)( AF488/CD3); and Alexa Fluor® 647-conjugated Fab Goat Anti-Mouse IgG1, Fcγ fragment specific was complexed with Mouse Anti-Human CD19 (BD #555410)(AF647/CD19).

Cells were incubated with complexed antibodies under various staining conditions for 30 minutes, washed, and analyzed on a BD FACSCalibur, gated on lymphocytes.


1. Cells incubated with AF488/CD3 and washed. A large population of T cells is visible in LR quadrant.

Left figure 1.0 μg anti-CD3/tube, right figure 0.5 μg anti-CD3/tube.


2. Cells incubated with AF647/CD19 and washed. A smaller population of B cells is visible in UL quadrant.

Left figure 1.0 μg anti-CD19/tube, right figure 0.1 μg anti-CD19/tube.


3. Cells incubated with a cocktail of AF488/CD3 and AF647/CD19 and washed.

Left figure: Anti-CD3 and anti-CD19 both at 1 μg of antibody/tube. Labeled Fab fragments are able to bind to incorrect primary antibody, resulting in unacceptable cross-talk. Most notably, excess AF647-FabuLight has bound to anti-CD3 on the T cells, shifting the T cell population into UR quadrant.
Right figure: Anti-CD3 is at 0.5 μg/tube, anti-CD19 is at 0.1 μg/tube. Titrated antibodies still result in cross-talk, though T cells have remained in the LR quadrant with insignificant loss of signal.


4. Cells incubated with AF647/CD19 (primary to less abundant target used first), washed, then incubated with AF488/CD3 and washed. Note specific labeling of T cells in LR quadrant and B cells in UL quadrant.

Left figure: Anti-CD3 and anti-CD19 both at 1 μg/tube. Right figure: Anti-CD3 is at 0.5 μg/tube, anti-CD19 is at 0.1 μg/tube. Titrated antibodies produce cleaner specific labeling with insignificant signal differences.


5. Cells incubated with AF488/CD3 (primary to more abundant target used first), washed, then incubated with AF647/CD19 and washed.

Left figure: Anti-CD3 and anti-CD19 both at 1 μg/tube. Excess AF647-FabuLight has bound to anti-CD3 on the T cells, shifting the population into UR quadrant.
Right figure: Anti-CD3 is at 0.5 μg/tube, anti-CD19 is at 0.1 μg/tube. T cells have shifted down. Further titration of anti-CD19 can improve this result, but labeling the less abundant primary first (as in #4) avoids the problem.