Next-generation hybridization chain reaction tools with enhanced sensitivities to detect challenging targets

Compared to traditional enzyme-based in situ amplification methods, Hybridization Chain Reaction v3.0 (HCR v3.0) offers high specificity for spatial RNA visualization but lacks the sensitivity needed for short or low-abundance targets, especially in thick tissue with high autofluorescence. We describe next-generation HCR detection methods that combine the specificity of HCR v3.0 with enzyme-based signal amplification through catalysis (HCR-Cat) or immunostaining (HCR-Immuno, HCR-Multi). These methods enhance sensitivity for robust spatial detection of both short and low-abundance targets, work well in challenging tissue environments, and enable broad utility across basic research and translational applications. These methods allow spatial detection of challenging targets that are poorly-accessible using HCR v3.0, as well as quantitative analysis of single transcripts even when targeting short RNAs with a limited number of probes.