Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping
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 detecti...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/16/8644 |
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| author | Thai Pham Christopher D. Nazaroff Joshua Labaer Jia Guo |
| author_facet | Thai Pham Christopher D. Nazaroff Joshua Labaer Jia Guo |
| author_sort | Thai Pham |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T08:45:17Z |
| format | Article |
| id | doaj.art-e7d377684e714ccc995a781ed9cd10c4 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T08:45:17Z |
| publishDate | 2021-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-e7d377684e714ccc995a781ed9cd10c42023-11-22T07:58:19ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-08-012216864410.3390/ijms22168644Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody StrippingThai Pham0Christopher D. Nazaroff1Joshua Labaer2Jia Guo3Biodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USABiodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USABiodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USABiodesign Institute & School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USAMultiplexed 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.https://www.mdpi.com/1422-0067/22/16/8644immunofluorescenceimmunohistochemistrysingle-cellin situ analysisproteomicsexpression heterogeneity |
| spellingShingle | Thai Pham Christopher D. Nazaroff Joshua Labaer Jia Guo Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping International Journal of Molecular Sciences immunofluorescence immunohistochemistry single-cell in situ analysis proteomics expression heterogeneity |
| title | Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping |
| title_full | Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping |
| title_fullStr | Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping |
| title_full_unstemmed | Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping |
| title_short | Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping |
| title_sort | ultrasensitive and multiplexed protein imaging with cleavable fluorescent tyramide and antibody stripping |
| topic | immunofluorescence immunohistochemistry single-cell in situ analysis proteomics expression heterogeneity |
| url | https://www.mdpi.com/1422-0067/22/16/8644 |
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