Stripping Multiplexed single-cell proteomic analysis with preserved spatial information of cells pledges great understanding toward the compositions, interactions, and functions of different cell types in complex biological systems. Nevertheless, it is restricted due to technical demands and low detection sensitivity from the existing multi-proteomic imaging technologies. To resolve the problem, we developed an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and Cleavable Fluorescent Tyramide (CFT). During each cycle of this method, protein targets were recognized by horseradish peroxidase-labeled antibodies, which catalyzed the covalent deposition of CFTs on or near the protein targets. After imaging, the fluorophores were chemically cleaved, and the antibodies were removed through the process of antibody striping. By continuously repeating cycles of staining, imaging, fluorophore cleavage, and antibody stripping, a large quantity of proteins can be quantified in every single cell in situ. Using this method, 20 proteins were analyzed in each of the 67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. From the distinctive protein expression profile and the microenvironment of these individual cells, they were further partitioned various cell clusters. Cell-cell interactions in the tissue sample was also discovered by examining which specific cell cluster are selectively associating or avoiding each other.

Ultrasensitive and multiplexed protein imaging with cleavable fluorescent tyramide and antibody

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.