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 step-by-step protocol describes tissue preparation, fixation, pretreatment, probe hybridization, signal amplification, 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.
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Acknowledgments
Supported in part by grants from the NIH (R43/44CA122444 to Y.L.) and the Department of Defense (Breast Cancer Research Program grant W81XWH-06-1-0682 to Y.L.).
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Wang, H. et al. (2015). Multiplex Fluorescent RNA In Situ Hybridization Via RNAscope. In: Hauptmann, G. (eds) In Situ Hybridization Methods. Neuromethods, vol 99. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2303-8_21
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DOI: https://doi.org/10.1007/978-1-4939-2303-8_21
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