Project Summary Comprehensive in situ protein profiling in individual cells of intact tissues holds great promise to unlock major mysteries in biology and medicine, since it can reveal the spatial organization, gene expression regulation, and interactions of the diverse cell types in multicellular organisms, such as brain tissues, solid tumors and developing embryos. However, this dream remains largely unrealized as the current multiplexed protein imaging methods are limited by their low detection sensitivity, sample throughput and multiplexing capacity. These fundamental limitations of the existing methods hinder their applications to profile formalin- fixed paraffin-embedded (FFPE) tissues, which are the most common type of preserved clinical samples. Our overarching goal is to develop a highly sensitive and high-throughput approach for comprehensive protein profiling in FFPE tissues in situ. To achieve this goal, we propose the following three specific aims. Aim 1: develop cleavable fluorescent antibodies for high-throughput and highly multiplexed protein profiling in FFPE tissues. Aim 2: develop highly sensitive probes for comprehensive protein profiling in FFPE tissues. Aim 3: classify the diverse cell types in human normal and Alzheimer’s brains by single-cell comprehensive protein profiling. If successful, these cleavable probes based technologies will have broad impact on understanding, diagnosis and treatment of complex diseases, such as neurodegenerative diseases and cancer. By mapping regulatory networks within individual cells and interrogating interactions among cells in a tissue, we will enhance our understanding of the molecular mechanisms of diseases, and may discover novel drug targets for more effective cellular targeted therapy. By pinpointing alterations in the abundances and positions of various proteins in single cells, new biomarkers can be identified to transform molecular diagnosis and treatment monitoring. The methods developed in the project can be readily adopted by other labs, and will be made publicly available.