title: A modified protocol for the detection of three different mRNAs with a new-generation in situ hybridization chain reaction on frozen sections.
abstract: A new multiple fluorescence in situ hybridization method based on hybridization chain reaction was recently reported, enabling simultaneous mapping of multiple target mRNAs within intact zebrafish and mouse embryos. With this approach, DNA probes complementary to target mRNAs trigger chain reactions in which metastable fluorophore-labeled DNA hairpins self-assemble into fluorescent amplification polymers. The formation of the specific polymers enhances greatly the sensitivity of multiple fluorescence in situ hybridization. In this study we describe the optimal parameters (hybridization chain reaction time and temperature, hairpin and salt concentration) for multiple fluorescence in situ hybridization via amplification of hybridization chain reaction for frozen tissue sections. The combined use of fluorescence in situ hybridization and immunofluorescence, together with other control experiments (sense probe, neutralization and competition, RNase treatment, and anti-sense probe without initiator) confirmed the high specificity of the fluorescence in situ hybridization used in this study. Two sets of three different mRNAs for oxytocin, vasopressin and somatostatin or oxytocin, vasopressin and thyrotropin releasing hormone were successfully visualized via this new method. We believe that this modified protocol for multiple fluorescence in situ hybridization via hybridization chain reaction would allow researchers to visualize multiple target nucleic acids in the future.
authors.affiliation: Department of Neurobiology, Key Laboratory of Molecular Neurobiology, Ministry of Education, Second Military Medical University, Shanghai, 200433, People's Republic of China.
authors.forename: Qian-Qian
authors.initials: QQ
authors.lastname: Sui
authors.affiliation: Department of Neurobiology, Key Laboratory of Molecular Neurobiology, Ministry of Education, Second Military Medical University, Shanghai, 200433, People's Republic of China.
authors.forename: Jiao
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Department of Anesthesiology, Changzheng Hospital, Second Military Medical University, Shanghai, 200433, People's Republic of China.|Department of Anesthesiology, The Third People's Hospital of Yancheng, Yancheng, 224001, People's Republic of China.
authors.forename: Xiangnan
authors.initials: X
authors.lastname: Li
authors.affiliation: Autonomic Neuroscience Centre, University College Medical School, Rowland Hill Street, London, NW3 2PF, UK.
authors.forename: Gillian E
authors.initials: GE
authors.lastname: Knight
authors.affiliation: Department of Neurobiology, Key Laboratory of Molecular Neurobiology, Ministry of Education, Second Military Medical University, Shanghai, 200433, People's Republic of China.
authors.forename: Cheng
authors.initials: C
authors.lastname: He
authors.affiliation: Autonomic Neuroscience Centre, University College Medical School, Rowland Hill Street, London, NW3 2PF, UK.|Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, VIC, Australia.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Burnstock
authors.affiliation: Department of Anesthesiology, Changzheng Hospital, Second Military Medical University, Shanghai, 200433, People's Republic of China.
authors.forename: Hongbin
authors.initials: H
authors.lastname: Yuan
authors.affiliation: Department of Neurobiology, Key Laboratory of Molecular Neurobiology, Ministry of Education, Second Military Medical University, Shanghai, 200433, People's Republic of China. zhxiang@hotmail.com.
authors.forename: Zhenghua
authors.initials: Z
authors.lastname: Xiang
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D005629:Frozen Sections; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats
chemical_list: D012333:RNA, Messenger
keywords: Brain section; Hybridization chain reaction; Multiple fluorescence in situ hybridization