Immunofluorescence is a laboratory technique extensively used in almost all fields of biology. The traditional way to carry out immunofluorescence is to apply an unlabeled primary antibody to recognize the target biomolecules, and then the fluorophore-labeled second antibody will be used to visualize the complex of the primary antibody and its target. Hence, traditional immunofluorescence is also defined as indirect immunofluorescence. In contrast, a method called direct immunofluorescence is that the primary antibody specific for the target molecule is labeled and visualized directly without the help of a secondary antibody. As many labeled secondary antibodies are commercially available and their assay signals are intense, the indirect immunofluorescence method was used more frequently than direct immunofluorescence.
Notably, along with the development of multiplex immunofluorescence, multiplexed panels are growing significantly, and the secondary antibody cross-reactivities have made panel design increasingly tricky. Secondary antibodies raised against immunoglobulins in one given species may also recognize immunoglobulins in other species through certain shared structures. This cross-reactivity causes background tissue staining or signal from non-target primary antibodies used in the same experiment, resulting in false-positive results and misinterpretations. To overcome the problem of secondary antibody cross-reactivities, many researchers attempt to avoid the utilization of secondary antibodies and only engage labeled primary antibodies in their experiments if possible.
In addition to avoiding non-specific binding with labeled secondary antibodies, directly using labeled primary antibodies simplified the multiplexing workflows significantly. In brief, traditional immunostaining includes incubations of primary and secondary antibodies and several rounds of washes in between. Applying labeled primary antibodies in multiplex immunofluorescence minimizes the number of incubation steps and wash steps dramatically. This simplification not only decreases hands-on time but also provides better data quality because fewer protocol steps and reagents used in one assay would diminish the interference of uncertain variables.
At last, antibody selection is always a pain for one given multiplex immunofluorescence. Especially when the traditional method (indirect immunofluorescence) is engaged in immunostaining and many antigens of interest are involved, the immunostaining panel design will be a nightmare. As the indirect immunofluorescence relies on secondary antibodies to detect primary antibodies, the primary antibodies used in the same experiment must be raised in different host species or be of different isotypes to be specially recognized. Conceivably, the number of existing host species and isotypes is finite, and therefore the panel size of the immunofluorescence is limited due to this constraint. To break through the bottleneck and make the multiplexing target detection more flexible, one of the feasible solutions is to use labeled primary antibodies directly. To date, more and more primary antibodies labeled with various fluorophores have been available, providing researchers with incredible flexibility.
In summary, multiplex immunofluorescence can be greatly simplified by covalently attaching the label directly to the primary antibody. Some of the advantages of direct assays are listed here:
1) Avoidance of non-specific binding with labeled secondary antibodies.
2) Reduction in the number of incubation steps.
3) Reduction in the number of wash steps.
4) Ability to multiplex with antibodies from the same species
5) Better data quality/less variability because of assay simplification.