HCR hairpin
HCR Citation Notes

For citation, please select from the list below as appropriate for your application:

  • 10-plex HCR Spectral Imaging
    HCR RNA-FISH/IF/PPI enable quantitative, high-resolution imaging of 10 RNA, protein, and/or protein:protein complex targets with 1-step HCR signal amplification for all targets simultaneously. The method is suitable for whole-mounts and delicate samples as it requires no repeated staining, imaging, registration, or stripping (Schulte et al., 2024b).
  • HCR PPI
    HCR protein:protein imaging (PPI) enables enzyme-free, multiplex, quantitative, high-resolution imaging of protein:protein complex targets in highly autofluorescent samples. HCR PPI is compatible with HCR RNA-FISH/IF for simultaneous imaging of RNA, protein, and protein:protein targets with 1-step HCR signal amplification performed for all target classes simultaneously (Schulte et al., 2024a).
  • HCR RNA-FISH/IF
    HCR RNA-FISH/IF provides a unified platform for multiplex, quantitative, high-resolution RNA and protein imaging in highly autofluorescent samples with 1-step HCR signal amplification performed for all RNA and protein targets simultaneously (Schwarzkopf et al., 2021).
  • HCR IF
    HCR immunofluorescence (IF) enables multiplex, quantitative, high-resolution protein imaging in highly autofluorescent samples (e.g., FFPE brain tissue sections) (Schwarzkopf et al., 2021).
  • HCR RNA-FISH
    • Third-generation HCR RNA-FISH (v3.0) enables multiplex, quantitative, high-resolution RNA fluorescence in situ hybridization (RNA-FISH) with automatic background suppression throughout the protocol for dramatically enhanced performance (signal-to-background, quantitative precision, single-molecule fidelity) and ease-of-use (no probe set optimization for new targets and organisms) (Choi et al., 2018).
    • Second-generation HCR RNA-FISH (v2.0) using DNA probes and amplifiers: 10× increase in signal, 10× reduction in cost, dramatic increase in reagent durability (Choi et al., 2014).
    • First-generation HCR RNA-FISH (v1.0) using RNA probes and amplifiers: multiplex RNA imaging in whole-mount vertebrate embryos with simultaneous signal amplification for up to 5 target RNAs (Choi et al., 2010).
  • Subcellular quantitative RNA, protein, and protein:protein imaging (qHCR imaging)
    HCR imaging enables analog relative quantitation of RNA, protein, and/or protein:protein complex targets (qHCR imaging) with subcellular resolution in the anatomical context of thick autofluorescent samples (e.g., whole-mount vertebrate embryos) (Trivedi et al., 2018; Choi et al., 2018; Schwarzkopf et al., 2021; Schulte et al., 2024a).
  • Single-molecule quantitative RNA imaging (dHCR imaging)
    HCR imaging enables digital RNA absolute quantitation (dHCR RNA imaging) with single-molecule resolution in the anatomical context of thick autofluorescent samples (e.g., 0.5 mm adult mouse brain sections) (Shah et al., 2016; Choi et al., 2018).
  • Read-out/read-in analysis framework
    The read-out/read-in analysis framework enables bidirectional quantitative discovery in an anatomical context (Trivedi et al., 2018).
  • Protocols in diverse sample types
    Protocols for HCR RNA-FISH, IF, and PPI in diverse sample types are adapted from the zoo paper (Choi et al., 2016):
    • bacteria in suspension
    • FFPE human tissue sections
    • generic sample in suspension
    • generic sample on a slide
    • mammalian cells in suspension
    • mammalian cells on a slide
    • whole-mount chicken embryos
    • whole-mount fruit fly embryos
    • whole-mount mouse embryos
    • whole-mount nematode larvae
    • whole-mount sea urchin embryos
    • whole-mount zebrafish embryos and larvae
  • HCR RNA flow cytometry
    HCR RNA flow cytometry enables analog RNA relative quantitation for high-throughput expression profiling of mammalian cells and bacteria without the need to engineer reporter lines (Choi et al., 2018).
  • HCR northern blots
    Simultaneous quantification of RNA target size and abundance with signal amplification for up to 5 target RNAs (Schwarzkopf & Pierce, 2016).
  • HCR mechanism
    The hybridization chain reaction (HCR) mechanism enables multiplex, quantitative, 1-step, isothermal, enzyme-free signal amplification in diverse technological settings (Dirks & Pierce, 2004).
HCR Technology References
  • Choi, H.M.T., Beck, V.A., & Pierce, N.A. (2014). Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability. ACS Nano, 8(5):4284-4294. (pdf, supp info, movie)
  • Choi, H.M.T., Calvert, C.R., Husain, N., Huss, D., Barsi, J.C., Deverman, B.E., Hunter, R.C., Kato, M., Lee, S.M., Abelin, A.C.T., Rosenthal, A.Z., Akbari, O.S., Li, Y., Hay, B.A., Sternberg, P.W., Patterson, P.H., Davidson, E.H., Mazmanian, S.K., Prober, D.A., van de Rijn, M., Leadbetter, J.R., Newman, D.K., Readhead, C., Bronner, M.E., Wold, B., Lansford, R., Sauka-Spengler, T., Fraser, S.E., & Pierce, N.A. (2016). Mapping a multiplexed zoo of mRNA expression. Development, 143:3632-3637. (pdf, supp info, fruit fly movie, sea urchin movie, zebrafish movie, chicken movie, mouse movie)
  • Choi, H.M.T., Schwarzkopf, M., Fornace, M.E., Acharya, A., Artavanis, G., Stegmaier, J., Cunha, A., & Pierce, N.A. (2018). Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development, 145, dev165753. (pdf, supp info, Dot Analysis 1.0 package)
  • Dirks, R.M., & Pierce, N.A. (2004). Triggered amplification by hybridization chain reaction. Proc Natl Acad Sci USA, 101(43), 15275–15278. (pdf)
  • Schulte, S.J., Shin, B., Rothenberg, E.V., & Pierce, N.A. (2024a). Multiplex, quantitative, high-resolution imaging of protein:protein complexes via hybridization chain reaction. ACS Chem Biol, 19(2), 280–288. (pdf, supp info)
  • Schwarzkopf, M., Liu, M. C., Schulte, S. J., Ives, R., Husain, N., Choi, H. M. T., & Pierce, N. A. (2021). Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization. Development, 148(22), dev199847. (pdf, supp info)
  • Schwarzkopf, M., & Pierce, N.A. (2016). Multiplexed miRNA northern blots via hybridization chain reaction. Nucleic Acids Res, 44(15), e129.
    (pdf, supp info)
  • Shah, S., Lubeck, E., Schwarzkopf, M., He, T.-F., Greenbaum, A., Sohn, C.H., Lignell, A., Choi, H.M.T., Gradinaru, V., Pierce, N.A., & Cai, L. (2016). Single-molecule RNA detection at depth via hybridization chain reaction and tissue hydrogel embedding and clearing. Development, 143:2862-2867. (pdf, supp info, movie 1, movie 2, movie 3)