3D RNA Detection in Millimeter-Scale Tissue Using RNAscope HiPlex.

Three-dimensional histology (3D) allows detailed visualization of molecular and cellular features within intact tissues, preserving spatial context that is often lost in traditional section-based approaches. However, this approach presents technical challenges, including tissue opacity and limited reagent penetration. While many tissue clearing techniques have been developed, only a handful are compatible with the detection of RNA targets, using RNA in situ hybridization. Here, we describe a protocol to apply the RNAscope HiPlex technology to tissue samples on the scale of a few cubic millimeters, combined with a solvent-based clearing method that permits 3D imaging. The workflow is demonstrated on mouse dorsal root ganglia, 2 mm-thick mouse brain sections, and human glioblastoma tissues.