The emerging field of 3D histology centers on the molecular profiling of intact organs for a comprehensive understanding of biological systems. While current methods focus largely on protein visualization, techniques for whole-organ RNA transcript imaging remain underdeveloped. Here we present a detailed protocol for Tris buffer-mediated retention of in situ hybridization chain reaction signal in cleared organs (TRISCO), a method for single-cell RNA three-dimensional mapping across tissue volumes. By ensuring homogeneous and well-preserved labeling throughout the entire tissue, TRISCO has been successfully applied to several mouse organs, including the brain, lung, heart, kidney and spinal cord, as well as to rat and guinea pig brains. The protocol is straightforward, is adaptable to diverse laboratory setups and research questions and avoids complex instrumentation, specialized expertise or harsh chemical treatments that might limit its applicability. The workflow, which can be completed in 10-15 days, includes flexible stopping points for convenient scheduling and is suitable for users with expertise in light-sheet microscopy and animal handling.
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