Methodological framework for chromogenic mRNA detection using in situ hybridization chain reaction.

In situ hybridization chain reaction (HCR) is a highly sensitive method for visualizing single-molecule messenger RNA (mRNA) using self-assembling hairpin DNA pairs. However, conventional in situ HCR is primarily optimized for fluorescence detection, limiting its applicability in routine pathology and in tissues with strong autofluorescence. In this study, we established a chromogenic in situ HCR protocol using short hairpin DNA that achieves sensitivity comparable to fluorescent in situ HCR. To achieve efficient amplification and avoid steric hindrance, we adopted an indirect labeling strategy in which hairpin DNA conjugated with haptens (biotin, digoxigenin, or fluorescein) is detected using enzyme-conjugated streptavidin or antibodies. We validated this approach across various mouse tissues, including the brain, kidney, and liver, and on both slide-mounted and free-floating sections. The protocol enabled the clear visualization of low-abundance transcripts such as Oxtr and Esr1. Furthermore, we demonstrated the versatility of this approach by performing duplex chromogenic staining for two mRNA targets and by combining in situ HCR with immunohistochemistry to visualize mRNA and protein simultaneously. This chromogenic in situ HCR retains the high sensitivity of HCR and adds the practical advantages of bright-field detection, thereby expanding the utility of in situ HCR in histological research.