Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

Abstract

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell–cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

Ultrasensitive and multiplexed protein imaging with clickable and cleavable fluorophores

“…We then quantified and compared the cleavage efficiency of 1-linker, 2-linker, and 3-linker CFTz (Figure 4C). 1-linker CFTz has the cleavage efficiency of ∼95.5%, which is consistent with our previously developed cleavable fluorescent probes (12, 17-20). With multiple cleavage sites, 2-linker, and 3-linker CFTz increased the cleavage efficiency to ∼97.5%.…”

“…Thus, it will not lead to false positive signals in subsequent protein quantification. Additionally, as documented in our previous studies, the TCEP, PTA and antibody stripping treatments will not damage the integrity of epitopes (19, 20). Therefore, the CFTz with the improved cleavage efficiency should enable a large number of varied proteins to be accurately quantified in single cells in situ.…”

“…The number of imaging cycles and the number of proteins quantified in each cycle are the key factors determining the multiplexing capacity of our approach. We have previously reported that our signal erasing treatment and antibody stripping process did not cause any damages to the integrity of the epitopes (19, 20). Here, we have demonstrated that at least 28 reiterative cycles can be carried out on the same tissue.…”

“…To enable highly sensitive and multiplexed protein imaging with unconjugated primary antibodies, our laboratory recently developed a spatial proteomic assay using cleavable fluorescent tyramide (CFT) (20). In this approach, protein targets are labeled with off-the-shelf primary antibodies.…”

Multiplexed Single-Cell in Situ Protein Profiling

“…To enable highly sensitive and multiplexed protein in situ analysis, an approach using cleavable fluorescent tyramide has also been developed. − In this method (Figure ), antibodies conjugated with horseradish peroxidase (HRP) are applied to stain the protein targets. HRP can enzymatically catalyze the coupling reaction between cleavable fluorescent tyramide and the tyrosine moieties on the antibodies or the endogenous proteins close to the targets.…”