Highly sensitive in situ proteomics with cleavable fluorescent tyramide reveals human neuronal heterogeneity

The ability to comprehensively profile proteins in intact tissues in situ is crucial for our understanding of health and disease. However, the existing methods suffer from low sensitivity and limited sample throughput. To address these issues, here we present a highly sensitive and multiplexed in situ protein analysis approach using cleavable fluorescent tyramide and off-the-shelf antibodies. Compared with the current methods, this approach enhances the detection sensitivity and reduces the imaging time by 1-2 orders of magnitude, and can potentially detect hundreds of proteins in intact tissues at the optical resolution. Applying this approach, we studied protein expression heterogeneity in genetically identical cells, and performed expression correlation analysis to identify coregulated proteins. We also profiled >6000 neurons in human formalin-fixed paraffin-embedded (FFPE) hippocampus. By partitioning these neurons into varied cell clusters based on their protein expression profiles, we observed different subregions of the hippocampus consist of neurons from distinct clusters.