Jackson ImmunoResearch Europe, Ltd

Multiple Labeling (ML) Using Labeled
Secondary Antibodies

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tech@jireurope.com tech@jacksonimmuno.com

Simultaneous detection of more than one antigen depends on at least two important criteria:

availability of secondary antibodies that (a) are derived from the same host species so that they do not recognise one another, (b) do not recognise (cross-react with) other primary antibodies used in the assay system, (c) do not recognise (cross-react with) immunoglobulins from other species possibly present in the assay system, and (d) do not cross-react with endogenous immunoglobulins possibly present in the tissues or on the surface of cells under investigation.
use of probes (enzyme-reaction products, fluorophores, or electron-dense particles) that are well resolved.

The affinity-purified antibodies marked ML (multiple labeling) have been specifically prepared to meet these criteria. One example of many possible multiple-labeling protocols using these reagents is shown in the following.

Mouse tissue antigen A	Mouse tissue antigen B	Mouse tissue antigen C
Step 1: 5% N. Donkey Serum Step 2: Goat Anti-Mouse antigen A Step 3: Probe 1-conjugated Donkey Anti-Goat IgG (H+L) (min X Ck, GP, Sy Hms, Hrs, Hu, Ms, Rb, Rat Sr Prot)	Step 4: 5% N. Donkey Serum Step 5: Rabbit Anti-Mouse antigen B Step 6: Probe 2-conjugated Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot)	Step 7: 5% N. Donkey Serum Step 8: Rat Anti-Mouse antigen C Step 9: Probe 3-conjugated Donkey Anti-Rat IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rb, Shp Sr Prot)

Note: Wash thoroughly after each step, including after the first blocking step. Do not dilute any antibody with normal serum.

In this example, the secondary antibodies used in Steps 3, 6, and 9 do not recognise each other since they are all made in donkeys. They have been solid-phase adsorbed so that they do not recognise the other primary antibodies used in Steps 2, 5, and 8. Also, they do not react with possible endogenous mouse immunoglobulins which may be present in the mouse tissues. For a review of multi-color immunofluorescence labeling with confocal microscopy see Brelje, Wessendorf, and Sorenson, "Multi-color laser scanning confocal immunofluorescence microscopy: Practical application and limitations." In "Cell Biological Applications of Confocal Microscopy". B. Matsumoto, ed. in Methods in Cell Biology. 1993. Academic Press, Inc., Orlando, Florida.