Ultrasensitive and Multiplexed Protein Imaging with Clickable and Cleavable Fluorophores

Single-cell spatial proteomic analysis holds great promise to advance our understanding of the composition, organization, interaction, and function of the various cell types in complex biological systems. However, the current multiplexed protein imaging technologies suffer from low detection sensitivity, limited multiplexing capacity, or are technically demanding. To tackle these issues, here, we report the development of a highly sensitive and multiplexed in situ protein profiling method using off-the-shelf antibodies. In this approach, the protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and cleavable fluorophores via click chemistry. Through repeated cycles of target staining, fluorescence imaging, and fluorophore cleavage, many proteins can be profiled in single cells in situ. Applying this approach, we successfully quantified 28 different proteins in human formalin-fixed paraffin-embedded (FFPE) tonsil tissue, which represents the highest multiplexing capacity among the tyramide signal amplification (TSA) methods. Based on their unique protein expression patterns and their microenvironment, ∼820,000 cells in the tissue are classified into distinct cell clusters. We also explored the cell–cell interactions between these varied cell clusters and observed that different subregions of the tissue are composed of cells from specific clusters.