Highly Sensitive and Multiplexed Protein Imaging With Cleavable Fluorescent Tyramide Reveals Human Neuronal Heterogeneity

Liao, Renjie; Mondal, Manas; Nazaroff, Christopher D.; Mastroeni, Diego; Coleman, Paul D.; LaBaer, Joshua; Guo, Jia

doi:10.3389/fcell.2020.614624

Cited by 11 publications

(16 citation statements)

References 73 publications

(98 reference statements)

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“…To enable highly sensitive and multiplexed protein imaging with off-the-shelf antibodies, our group developed a reiterative protein staining approach using cleavable fluorescent tyramide (CFT) [17]. We demonstrated that its sensitivity is improved by about two orders of magnitude compared with other existing methods.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Introductionmentioning

confidence: 99%

“…Additionally, with tris(2-carboxyethyl) phosphine (TCEP) as the signal removal reagent, the first-generation CFT requires 65 • C to remove~95% of the staining signals. Therefore, this relatively high reaction temperature could damage the integrity of the epitopes [17].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…Without signal amplification, the low detection sensitivity of these methods limits their applications to study low-expression proteins or to examine specimens with high autofluorescence, such as formalin-fixed paraffin-embedded (FFPE) tissues [18]. To tackle these issues, several laboratories, including ours, have developed several sensitive and multiplexed protein imaging technologies by signal amplifications with biotin-streptavidin interaction [19], oligonucleotide hybridization [20], and horseradish peroxidase (HRP) [21,22]. However, these methods require a chemical-, oligonucleotide-or HRP-labeled primary antibodies to recognize the protein targets.…”

Section: Introductionmentioning

confidence: 99%

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

Pham

Nazaroff

LaBaer

et al. 2021

IJMS

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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.

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“…To assess the fluorophore cleavage efficiency, we stained mRNA PPIB in an FFPE mouse lung tissue using tyramide-N 3 -Cy5 ( Figure 1 D). This CFT molecule has been recently developed in our laboratory for multiplexed protein imaging [ 27 ]. We have demonstrated that tyramide-N 3 -Cy5 can be successfully converted by HRP to a short-lived reactive radical.…”

Section: Resultsmentioning

confidence: 99%

“…Integrated in situ analysis of RNA and proteins in the same specimen is of increasing importance in studies of gene expression regulation [ 29 ] and disease diagnosis [ 30 ]. Our laboratory recently developed cleavable fluorescent probes [ 27 , 31 , 32 , 33 ] for multiplexed protein imaging and demonstrated that these probes enable a large amount of different proteins to be accurately quantified in their native cellular contexts in single cells. To evaluate whether the multiplexed RNA and proteins in situ analysis can be combined in the same tissue using CFT, we stained two proteins and five mRNA sequentially using tyramide-N 3 -Cy5 in a mouse spinal cord tissue ( Figure 7 A).…”

Section: Resultsmentioning

confidence: 99%

Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide

Lü

LaBaer

Guo

2021

Cells

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Understanding the composition, regulation, and function of complex biological systems requires tools that quantify multiple transcripts at their native cellular locations. However, the current multiplexed RNA imaging technologies are limited by their relatively low sensitivity or specificity, which hinders their applications in studying highly autofluorescent tissues, such as formalin-fixed paraffin-embedded (FFPE) tissues. To address this issue, here we develop a multiplexed in situ RNA profiling approach with a high sensitivity and specificity. In this approach, transcripts are first hybridized by target-specific oligonucleotide probes in pairs. Only when these two independent probes hybridize to the target in tandem will the subsequent signal amplification by oligonucleotide hybridization occur. Afterwards, horseradish peroxidase (HRP) is applied to further amplify the signal and stain the target with cleavable fluorescent tyramide (CFT). After imaging, the fluorophores are chemically cleaved and the hybridized probes are stripped by DNase and formamide. Through cycles of RNA staining, fluorescence imaging, signal cleavage, and probe stripping, many different RNA species can be profiled at the optical resolution. In applying this approach, we demonstrated that multiplexed in situ RNA analysis can be successfully achieved in both fixed, frozen, and FFPE tissues.

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Highly Sensitive and Multiplexed Protein Imaging With Cleavable Fluorescent Tyramide Reveals Human Neuronal Heterogeneity

Cited by 11 publications

References 73 publications

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

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

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

Highly Sensitive and Multiplexed In Situ RNA Profiling with Cleavable Fluorescent Tyramide

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