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Multiplexed in situ protein imaging using DNA-barcoded antibodies with extended hybridization chain reactions

View ORCID ProfileYu Wang, Yitian Zeng, View ORCID ProfileSinem K. Saka, Wenxin Xie, Isabel Goldaracena, Richie E. Kohman, Peng Yin, George M. Church
doi: https://doi.org/10.1101/274456
Yu Wang
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA
2Department of Genetics, Harvard Medical School, Boston, USA
3Department of System Biology, Harvard Medical School, Boston, USA
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  • ORCID record for Yu Wang
Yitian Zeng
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA
2Department of Genetics, Harvard Medical School, Boston, USA
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Sinem K. Saka
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA
3Department of System Biology, Harvard Medical School, Boston, USA
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  • ORCID record for Sinem K. Saka
Wenxin Xie
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA
2Department of Genetics, Harvard Medical School, Boston, USA
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Isabel Goldaracena
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA
3Department of System Biology, Harvard Medical School, Boston, USA
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Richie E. Kohman
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA
2Department of Genetics, Harvard Medical School, Boston, USA
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Peng Yin
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA
3Department of System Biology, Harvard Medical School, Boston, USA
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George M. Church
1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, USA
2Department of Genetics, Harvard Medical School, Boston, USA
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  • For correspondence: gchurch@genetics.med.harvard.edu
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Abstract

Immunofluorescence (IF) imaging using antibodies to visualize specific biomolecules is a widely used technique in both biological and clinical laboratories. Standard IF imaging methods using primary antibodies followed by secondary antibodies have low multiplexing capability due to limited availability of primary antibodies raised in different animal species. Here, we used a DNA-based signal amplification method, Hybridization Chain Reaction (HCR), to replace secondary antibodies to achieve multiplexed imaging using primary antibodies of the same species with superior signal intensity. To enable imaging with DNA-conjugated antibodies, we developed a new antibody staining protocol to minimize nonspecific binding of antibodies caused by conjugated DNA oligonucleotides. We also expanded the HCR hairpin pool from previously published 5 to 13 for highly multiplexed in situ imaging. We finally demonstrated multiplexed in situ protein imaging using the technique in both cultured cells and mouse retina sections.

Competing Interest Statement

G.M.C. is a cofounder of Readcoor, Inc. and P.Y. is a cofounder of Utivue, Inc.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted May 10, 2020.
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Multiplexed in situ protein imaging using DNA-barcoded antibodies with extended hybridization chain reactions
Yu Wang, Yitian Zeng, Sinem K. Saka, Wenxin Xie, Isabel Goldaracena, Richie E. Kohman, Peng Yin, George M. Church
bioRxiv 274456; doi: https://doi.org/10.1101/274456
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Multiplexed in situ protein imaging using DNA-barcoded antibodies with extended hybridization chain reactions
Yu Wang, Yitian Zeng, Sinem K. Saka, Wenxin Xie, Isabel Goldaracena, Richie E. Kohman, Peng Yin, George M. Church
bioRxiv 274456; doi: https://doi.org/10.1101/274456

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