Padlock Probe-Initiated Hybridization Chain Reaction for In Situ RNA Imaging.

Spatially resolved RNA analysis is essential for understanding gene expression patterns in biological systems. RNA in situ hybridization is widely used but often suffers from insufficient sensitivity for low-abundance targets or excessive background from signal amplification. Here, we present α-HCR, a padlock probe-assisted hybridization chain reaction strategy for RNA in situ imaging. In α-HCR, padlock probes first hybridize to target RNA molecules, followed by probe circularization and hybridization chain reaction-mediated signal amplification. Stringent washing was applied to remove uncircularized probes before signal amplification, thereby minimizing nonspecific fluorescence. We demonstrated the feasibility of α-HCR in cultured cells, formalin-fixed paraffin-embedded (FFPE) mouse kidney sections, and whole-mount zebrafish embryos. In tissue sections, α-HCR generated spatial expression patterns comparable to those obtained using padlock probe-based rolling circle amplification assays. In intact zebrafish embryos, α-HCR enabled multiplexed RNA imaging across different tissue depths and showed performance comparable to conventional in situ HCR approaches. These results demonstrate α-HCR as a versatile method for spatial RNA detection in both sectioned tissues and intact 3D specimens, expanding the toolbox available for in situ transcriptomic analysis.