Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.
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.
Pinaud
Raphael
R
Department of Brain and Cognitive Sciences, University of Rochester, Rochester, New York 14627, USA. pinaud@bcs.rochester.edu
Mello
Claudio V
CV
Velho
Tarciso A
TA
Wynne
Ryan D
RD
Tremere
Liisa A
LA
D000107:Acetylation; D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D046369:Finches / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D023281:Genomics / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008867:Microtomy; D009475:Neurons, Afferent / Q000378:metabolism; D015347:RNA Probes / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry; D017423:Sequence Analysis, RNA / Q000379:methods
D015347:RNA Probes; D012333:RNA, Messenger