Multiplexed in situ protein profiling with high-performance cleavable fluorescent tyramide

Thai Pham, Renjie Liao, Joshua Labaer, Jia Guo

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.

Original languageEnglish (US)
Article number2206
JournalMolecules
Volume26
Issue number8
DOIs
StatePublished - 2021

Keywords

  • Heterogeneity
  • Immunofluorescence
  • Immunohistochemistry
  • Proteomics
  • Single-cell

ASJC Scopus subject areas

  • Analytical Chemistry
  • Chemistry (miscellaneous)
  • Molecular Medicine
  • Pharmaceutical Science
  • Drug Discovery
  • Physical and Theoretical Chemistry
  • Organic Chemistry

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