The datasets for Precise immunofluorescence canceling enables highly multiplexed imaging

Cell states are regulated by responsive signal pathways upon ligand-binding to the receptor and inter-cellular interactions. Thus, high-resolution imaging has been attempted to explore the dynamics of signaling. Recently, multiplexed imaging has been introduced to profile cell state by acquisition of comprehensive spatial protein information in the cells. However, it is still challenging to compromise resolution for visualizing activated signals. Here we developed ‘Precise Emission Canceling Antibody (PECAb)’ attached with erasable fluorescent labeling. PECAbs allow high-resolution sequential imaging using 206 antibodies and it allowed reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system serves as a comprehensive platform for analyzing complex cell processes.