High-Resolution Fluorescence In Situ Hybridization to Detect mRNAs in Neuronal Compartments In Vitro

The localization of specific mRNAs into dendrites and/or axons is an important mechanism to enrich �proteins at their sites of function and influence neuronal development, plasticity, and repair. The fluorescence in situ hybridization (FISH) methods described here have provided high sensitivity and resolution enabling investigation into the mechanism, regulation, and function of mRNA localization in vitro and in vivo. Two methods are described in detail. The first method employs digoxigenin- or fluorophore-conjugated oligonucleotide probes for the detection of localized mRNAs in dendrites, spines, axons, and growth cones of cultured neurons. The second method employs digoxigenin-labeled RNA probes and fluorescence tyramide amplification for the detection of less abundant mRNAs localized to dendrites in vivo. Both methods enable the visualization and quantification of mRNA granules, and changes in their localization in response to various stimuli. The high-resolution FISH technology described here has broader applications beyond the study of mRNA localization. It enables the quantitative analyses of developmental and cell type-specific patterns of gene expression, and how these are modified by physiological signals or during disease states.