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Published May 12, 2020 | public
Book Section - Chapter

Multiplexed Quantitative In Situ Hybridization with Subcellular or Single-Molecule Resolution Within Whole-Mount Vertebrate Embryos: qHCR and dHCR Imaging (v3.0)


In situ hybridization based on the mechanism of hybridization chain reaction (HCR) enables multiplexed quantitative mRNA imaging in the anatomical context of whole-mount vertebrate embryos. Third-generation in situ HCR (v3.0) provides automatic background suppression throughout the protocol, dramatically enhancing performance and ease of use. In situ HCR v3.0 supports two quantitative imaging modes: (1) qHCR imaging for analog mRNA relative quantitation with subcellular resolution in an anatomical context and (2) dHCR imaging for digital mRNA absolute quantitation with single-molecule resolution in an anatomical context. Here, we provide protocols for qHCR and dHCR imaging in whole-mount zebrafish, chicken, and mouse embryos.

Additional Information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature. First Online: 12 May 2020. We thank M.E. Fornace for developing the Dot Analysis 1.0 software package for analyzing dHCR images and V. Trivedi for developing the Read-out/Read-in 1.0 software package for performing read-out/read-in analyses on multiplexed qHCR images in vertebrate embryos. Within the Beckman Institute at Caltech, we thank the following for assistance: C.R. Calvert and G.J. Shin (Molecular Technologies), A. Collazo and S. Wilbert (Biological Imaging Facility), and J. Stegmaier and A. Cunha (Center for Advanced Methods in Image Analysis). This work was funded by the National Institutes of Health (National Institute of Biomedical Imaging and Bioengineering R01EB006192), by the Defense Advanced Research Projects Agency (HR0011-17-2-0008; the findings are those of the authors and should not be interpreted as representing the official views or policies of the US Government), by the Beckman Institute at Caltech (Programmable Molecular Technology Center, PMTC), by the Gordon and Betty Moore Foundation (GBMF2809), by the National Science Foundation Molecular Programming Project (NSF-CCF-1317694), by a Professorial Fellowship at Balliol College, University of Oxford, and by the Eastman Visiting Professorship at the University of Oxford. Competing Interests: The authors declare competing financial interests in the form of patents, pending patent applications, and a startup company (Molecular Instruments).

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