Sequentially‐Activated Antibodies Based on Programmable DNA Tags for Rapid Multiplexed Protein Imaging

Abstract The achievement of rapid multiplexed protein imaging is limited by the use of stimulating reagents, extensive incubating and washing steps, and the low fluorescence intensity of targets. In this study, sequentially‐activated DNA tags are developed and combined them with primary antibodies using signal enhancement strategies to create sequentially‐activated antibodies (SAAs). These SAAs enable rapid, wash‐free sequential imaging of different protein targets. The samples are pre‐processed to label all targets of interest with SAAs simultaneously, and the signal is turned ON for only one target in each stage. The sequential imaging of multiple targets is achieved through wash‐free strand displacement reactions that exhibit rapid kinetics with t_1/2 < 10 s in a cellular context. Remarkably, this method successfully demonstrates sequential imaging of nine different protein targets within just a few minutes. This all‐in‐one platform for multiplexed protein imaging holds great promise for diverse applications in immunofluorescence imaging.