Next Article in Journal
Catalytic Conversion of Xylose to Furfural by p-Toluenesulfonic Acid (pTSA) and Chlorides: Process Optimization and Kinetic Modeling
Previous Article in Journal
Identification of a Prenyl Chalcone as a Competitive Lipoxygenase Inhibitor: Screening, Biochemical Evaluation and Molecular Modeling Studies
Previous Article in Special Issue
Revisiting the Rate-Limiting Step of the ANS–Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity
Article

Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

Biodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: Thomas Gustavsson
Molecules 2021, 26(8), 2206; https://doi.org/10.3390/molecules26082206
Received: 28 February 2021 / Revised: 6 April 2021 / Accepted: 8 April 2021 / Published: 12 April 2021
(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)
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. View Full-Text
Keywords: single-cell; proteomics; immunofluorescence; immunohistochemistry; heterogeneity single-cell; proteomics; immunofluorescence; immunohistochemistry; heterogeneity
Show Figures

Figure 1

MDPI and ACS Style

Pham, T.; Liao, R.; Labaer, J.; Guo, J. Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide. Molecules 2021, 26, 2206. https://doi.org/10.3390/molecules26082206

AMA Style

Pham T, Liao R, Labaer J, Guo J. Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide. Molecules. 2021; 26(8):2206. https://doi.org/10.3390/molecules26082206

Chicago/Turabian Style

Pham, Thai, Renjie Liao, Joshua Labaer, and Jia Guo. 2021. "Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide" Molecules 26, no. 8: 2206. https://doi.org/10.3390/molecules26082206

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop