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Highly multiplexed in situ protein imaging with signal amplification by Immuno-SABER
Open accessPreprint — published as 10.1038/s41587-019-0207-y
Abstract
Probing the molecular organization of tissues requires in situ analysis by microscopy. However current limitations in multiplexing, sensitivity, and throughput collectively constitute a major barrier for comprehensive single-cell profiling of proteins. Here, we report Immunostaining with Signal Amplification By Exchange Reaction (Immuno-SABER), a rapid, highly multiplexed signal amplification method that simultaneously tackles these key challenges. Immuno-SABER utilizes DNA-barcoded antibodies and provides a method for highly multiplexed signal amplification via modular orthogonal DNA concatemers generated by Primer Exchange Reaction. This approach offers the capability to preprogram and control the amplification level independently for multiple targets without in situ enzymatic reactions, and the intrinsic scalability to rapidly amplify and image a large number of protein targets. We validated our approach in diverse sample types including cultured cells, cryosections, FFPE sections, and whole mount tissues. We demonstrated independently tunable 5-180-fold amplification for multiple targets, covering the full signal range conventionally achieved by secondary antibodies to tyramide signal amplification, as well as simultaneous signal amplification for 10 different proteins using standard equipment and workflow. We further combined Immuno-SABER with Expansion Microscopy to enable rapid and highly multiplexed super-resolution tissue imaging. Overall, Immuno-SABER presents an effective and accessible platform for rapid, multiplexed imaging of proteins across scales with high sensitivity.
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Metadata
- Authors
- Saka, S. K., Wang, Y., Kishi, J. Y., Zhu, A., Zeng, Y., Xie, W., Kirli, K., Yapp, C., Cicconet, M., Beliveau, B. J., Lapan, S. W., Yin, S., Lin, M., Boyden, E. S., Kaeser, P. S., Pihan, G., Church, G. M., & Yin, P.
- Affiliations
- Harvard University (US)
- MIT Media Lab (US)
- Beth Israel Deaconess Medical Center (US)
- Corresponding author
- Saka, Sinem K.
- Wang, Yu
- Yin, Peng
- ORCID
- Venue
- Nature Biotechnology
- Publisher
- openRxiv
- Published
- 2018-12-28
- Volume
- 37
- Issue
- 9
- Pages
- 1080-1090
- ISSN
- 0733-222X (ISSN-L)
- PMC
- PMC6728175
- OpenAlex
- W2968292465
- Cited by
- 485
- References
- 87
- Citations by year
- 2026: 58 · 2025: 74 · 2024: 81 · 2023: 61 · 2022: 87 · 2021: 68 · 2020: 48 · 2019: 7 · 2018: 1
- FWCI
- 17.37
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- cc-by-nc-nd
- Locations
- Nature Biotechnology (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
- Funders
- National Science Foundation (1317291)
- U.S. Department of Defense (N000141612410, N000141812549, N000141612182)
- Howard Hughes Medical Institute
- U.S. Department of Health and Human Services (R01NS083898, 1UG3HL145600/HuBMAP, 1U01MH106011, 1R01GM124401, R01MH113279, RM1 HG008525, 1R01EB018659, 1DP1GM133052, 5K99EY028215-02, 1R01MH113349)
- Damon Runyon Cancer Research Foundation
- Harvard Medical School
- National Institutes of Health (5k99ey028215-02, R01NS083898, RM1 HG008525, R01MH113279, NS072030, 1UG3HL145600/HuBMAP, 1UG3HL145600/HuBMAP, 1R01EB018659, 1U01MH106011, 1DP1GM133052, 1R01GM124401)
- Hansjörg Wyss Institute for Biologically Inspired Engineering, Harvard University
- National Heart, Lung, and Blood Institute (UG3HL145600)
- Office of Naval Research (N00014-16-1-2410, N00014-16-1-2182, N00014-18-1-2549, N00014-16-1-2410, (N00014-18-1-2549, N00014-16-1-2182, N00014)
- National Institute of Mental Health (U01MH106011, R01MH113349, R01MH113279)
- National Human Genome Research Institute (RM1HG008525)
- National Eye Institute (K99 EY028215)
- National Institute of General Medical Sciences (DP1GM133052, R01GM124401)
- National Institute of Neurological Disorders and Stroke (#NS072030, P30NS072030, R01NS083898)
- National Institute of Biomedical Imaging and Bioengineering (R01EB018659)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- primer exchange reaction, expansion microscopy, formalin-fixed paraffin-embedded sections, frozen section
- MeSH terms
- Animals, Antibodies, Cell Line, DNA, Fluorescent Dyes, Humans, Immunohistochemistry, Microscopy, Fluorescence, Proteins, Retina, Staining and Labeling, In Situ Hybridization, Fluorescence, Mice, DNA Barcoding, Taxonomic
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