smFISH: Quantifying and Mapping RNA at the Single-Molecule Level

RNA localization regulates spatial and temporal control of protein synthesis, influencing processes such as development, neuronal signaling, cell migration, and stress responses. Single-molecule fluorescence in situ hybridization (smFISH) enables direct visualization and quantification of individual transcripts within cells, providing both absolute counts and subcellular localization of RNAs. Here, we present a detailed, step-by-step protocol for smFISH in adherent mammalian cells, including probe design and labeling, sample preparation, hybridization, imaging, and analysis. This guide offers a reproducible workflow that facilitates the study of RNA localization and expression at single-molecule resolution.