Multiplex fluorescent RNA in situ hybridization via RNAscope

Abstract

Multiplex fluorescent in situ hybridization is a useful tool for neurobiology applications. We have developed a novel RNA ISH technology, RNAscope, with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. Here, we present a detailed protocol of Multiplex Fluorescent RNAscope Assay that permits visualization of three target genes simultaneously on fresh frozen brain tissue sections. The stepby- step protocol describes tissue preparation, fixation, pretreatment, probe hybridization, signal amplifi- cation, and visualization. The Multiplex Fluorescent RNAscope Assay is particularly useful for detecting low-copy genes, as it offers high sensitivity and specificity. We also discuss critical steps for ensuring successful experiments.