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HCR™ Technology Citation Notes

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

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  • 10-Plex HCR™ Spectral Imaging
    HCR™ RNA-FISH/IF enables quantitative high-resolution imaging of 10 RNA and/or protein targets with 1-step HCR™ signal amplification for all targets simultaneously. The method is suitable even for whole-mounts and delicate samples as it requires no repeated staining, imaging, registration, or stripping (Schulte et al., 2024).  

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  • HCR™ RNA-FISH/IF
    HCR™ RNA-FISH/IF enables a unified approach to multiplex, quantitative, high-resolution RNA fluorescence in situ hybridization (RNA-FISH) and protein immunofluorescence (IF), with quantitative 1-step enzyme-free HCR™ signal amplification performed for all RNA and protein targets simultaneously  (Schwarzkopf et al., 2021).  

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  • HCR™ IF
    HCR™ IF enables multiplex, quantitative, high-resolution protein immunofluorescence (IF) in highly autofluorescent samples (e.g., FFPE brain tissue sections) (Schwarzkopf et al., 2021).

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  • 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 HCR™ probes and DNA HCR™ 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 HCR™ probes and RNA HCR™ amplifiers: multiplex mRNA imaging in whole-mount vertebrate embryos with simultaneous signal amplification for up to 5 target mRNAs (Choi et al., 2010). ​

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  • Protocols in Diverse Sample Types
    Protocols for HCR™ RNA-FISH and/or IF in diverse sample types are adapted from the zoo paper (Choi et al., 2016):​  ​

    • bacteria in suspension

    • FFPE human tissue sections 

    • generic sample in solution

    • generic sample on a slide

    • mammalian cells on a slide

    • mammalian cells in suspension

    • 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

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  • 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 engineering reporter lines (Choi et al., 2018).     ​

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  • HCR™ Amplifiers
    HCR™ Amplifiers enable multiplex, quantitative, 1-step, isothermal, enzyme-free signal amplification in diverse technological settings (Dirks & Pierce, 2004). ​​

​HCR™ Technology References

  • Choi, H.M.T., Chang, J.Y., Trinh, L.A., Padilla, J.E., Fraser, S.E., & Pierce, N.A. (2010). Programmable in situ amplification for multiplexed imaging of mRNA expression. 
    Nat Biotechnol, 28:1208–1212.

  • 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. 

  • 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. 

  • 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. 

  • Dirks, R.M., & Pierce, N.A. (2004). Triggered amplification by hybridization chain reaction. Proc Natl Acad Sci USA, 101(43), 15275–15278. 

  • Schwarzkopf, M., & Pierce, N.A. (2016). Multiplexed miRNA northern blots via hybridization chain reaction. Nucleic Acids Res, 44(15), e129. 

  • 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. 

  • Schulte, S.J., Fornace, M.E., Hall, J.L., Shin, G.J., & Pierce, N.A. (2024).  HCR spectral imaging: 10-plex, quantitative, high-resolution RNA and protein imaging in highly autofluorescent samples. Development, 151 (4): dev202307.

  • 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. 

  • Trivedi, V., Choi, H.M.T., Fraser, S.E., & Pierce, N.A. (2018). Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos. Development, 145, dev156869. 

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