Abstract
Single-molecule fluorescence in situ hybridization (smFISH) allows spatial mapping of gene expression. This protocol presents advances in smFISH fidelity and flexibility in intact murine sensory nervous system tissue. An approach using RNAscope probes allows multiplexing, enhanced target specificity, and immunohistochemistry compatibility. Computational strategies increase quantification accuracy of mRNA puncta with a point spread function for clustered transcripts in the dorsal root ganglion and 3D masking for intermingled sciatic nerve cell types. Approaches are validated for mRNAs of modest (Lin28a) and medium (Ppib) steady-state abundance in neurons.