Abstract
Profiling molecular expression in situ allows the integration of biomolecular and cellular features, enabling an in-depth understanding of biological systems. Multiplexed immunofluorescence methods can visualize tens to hundreds of proteins from individual tissue samples, but their application is usually limited to thin tissue sections. Multiplexed immunofluorescence of thick tissues or intact organs will enable high-throughput profiling of cellular protein expression within 3D tissue architectures (e.g., blood vessels, neural projections, tumors), opening a new dimension in diverse biological research and medical applications. We will review current multiplexed immunofluorescence methods and discuss possible approaches and challenges to achieve 3D multiplexed immunofluorescence.