  Tissues and organs are composed of distinct cell types that must operate in
concert to perform physiological functions. Efforts to create high-dimensional
biomarker catalogs of these cells are largely based on transcriptomic
single-cell approaches that lack the spatial context required to understand
critical cellular communication and correlated structural organization. To
probe in situ biology with sufficient coverage depth, several multiplexed
protein imaging methods have recently been developed. Though these
antibody-based technologies differ in strategy and mode of immunolabeling and
detection tags, they commonly utilize antibodies directed against protein
biomarkers to provide detailed spatial and functional maps of complex tissues.
As these promising antibody-based multiplexing approaches become more widely
adopted, new frameworks and considerations are critical for training future
users, generating molecular tools, validating antibody panels, and harmonizing
datasets. In this perspective, we provide essential resources and key
considerations for obtaining robust and reproducible multiplexed antibody-based
imaging data compiling specialized knowledge from domain experts and technology
developers.
