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Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

By Harry M.T. Choi, Maayan Schwarzkopf, Mark E. Fornace, Aneesh Acharya, Georgios Artavanis, Johannes Stegmaier, Alexandre Cunha, Niles A. Pierce

Posted 19 Mar 2018
bioRxiv DOI: 10.1101/285213 (published DOI: 10.1242/dev.165753)

In situ hybridization based on the mechanism of hybridization chain reaction (HCR) has addressed multi-decade challenges to imaging mRNA expression in diverse organisms, offering a unique combination of multiplexing, quantitation, sensitivity, resolution, and versatility. Here, with third-generation in situ HCR, we augment these capabilities using probes and amplifiers that combine to provide automatic background suppression throughout the protocol, ensuring that even if reagents bind non-specifically within the sample they will not generate amplified background. Automatic background suppression dramatically enhances performance and robustness, combining the benefits of higher signal-to-background with the convenience of using unoptimized probe sets for new targets and organisms. In situ HCR v3.0 enables multiplexed quantitative mRNA imaging with subcellular resolution in the anatomical context of whole-mount vertebrate embryos, multiplexed quantitative mRNA flow cytometry for high-throughput single-cell expression profiling, and multiplexed quantitative single-molecule mRNA imaging in thick autofluorescent samples.

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