Complex Made Simple: Antibodies in Multiplexed Imaging

Antibodies can be used experimentally to detect proteins on an electrophoretic gel (immunoblotting). In addition, they can be deployed in color changing antigen recognition assays known as ELISAs (enzyme-linked immunosorbent assays) or can be directly conjugated to dyes and enzymes and used directly to stain a protein of interest in tissue. It is this last function, and other closely related methods, that has seen wide use in both traditional immunohistochemistry (IHC) and multiplexed imaging.

Multiplexed imaging opens the door to the use of many more biomarkers in a study than would be otherwise be practical using a traditional imaging approach. The “budget” of available antibodies is therefore much larger. However, care should be taken in the selection of antibodies to ensure the highest return on investment for the time and effort spent on an experiment.

The experimental biomarkers of immediate interest should first be considered. It may also be useful to identify biomarkers in similar or related biological pathways that may flesh out the understanding of an experiment. The ultimate method of image analysis should also be considered. If automated image analysis through image segmentation is employed, a variety of markers can be included in the study to outline cell compartments and tissues more clearly. It may also be useful to include biomarkers with broad staining patterns to assess tissue quality throughout the length of the study. Biomarkers that more clearly identify cellular subtypes, such as immune cell types, should also be considered to add depth to the study.

Identifying, sourcing, testing, and using antibodies for a multiplexed imaging study is a complex and often challenging process. Careful study design and validation processes will greatly aid the researcher in reliable, robust experiments.