Fluorescent in situ hybridization in primary hippocampal neurons to detect localized mRNAs

Abstract

In neurons, local translation of mRNAs contributes to axon outgrowth and activity-dependent synaptic plasticity. The identification and visualization of individual localized mRNAs is critical for understanding these processes. Here, we describe a sensitive fluorescence in situ hybridization (FISH) method that provides high-resolution information on mRNA localization using in vitro cultured rat hippocampal neurons. The method employs digoxigenin (DIG)-labeled RNA probes and fluorescent tyramide signal amplification for detection of mRNAs. It enables not only the visualization but also quantification of mRNA granules, and changes in their localization in response to various stimuli. The method is also compatible with immunocytochemistry, which allows for the co-visualization of RNAs and proteins in fixed cells.