Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization

Nature Protocols
Raphael PinaudLiisa A Tremere

Abstract

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.

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Citations

Aug 1, 2012·Proceedings of the National Academy of Sciences of the United States of America·Ho TsoiHo Yin Edwin Chan
May 29, 2010·Molecular Neurodegeneration·Bin MaNaoko Tanese
Aug 26, 2010·Journal of Visualized Experiments : JoVE·Jin Kwon JeongRaphael Pinaud
Aug 28, 2015·Nature Protocols·Takashi FujiiTsutomu Masujima
Oct 21, 2015·Tumour Biology : the Journal of the International Society for Oncodevelopmental Biology and Medicine·Lin SuXueyun Huo
May 1, 2012·Journal of Experimental Neuroscience·Liisa A TremereRaphael Pinaud
Nov 8, 2017·Journal of the American Society of Nephrology : JASN·Sepideh Sheybani-DelouiNorman D Rosenblum
Apr 26, 2013·Molecular BioSystems·Beena M Kadakkuzha, Sathyanarayanan V Puthanveettil

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