RT Journal Article SR Electronic T1 Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust JF bioRxiv FD Cold Spring Harbor Laboratory SP 285213 DO 10.1101/285213 A1 Harry M.T. Choi A1 Maayan Schwarzkopf A1 Mark E. Fornace A1 Aneesh Acharya A1 Georgios Artavanis A1 Johannes Stegmaier A1 Alexandre Cunha A1 Niles A. Pierce YR 2018 UL http://biorxiv.org/content/early/2018/03/19/285213.abstract AB 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.SUMMARY In situ hybridization chain reaction (HCR) v3.0 exploits automatic background suppression to enable multiplexed quantitative mRNA imaging and flow cytometry with dramatically enhanced ease-of-use and performance.