title: An optimized protocol for high-throughput in situ hybridization of zebra finch brain.
abstract: In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advanced nonradioactive ISH methods that are based on the use of digoxigenin (DIG)-labeled probes and chromogenic detection have better spatial resolution than emulsion autoradiography techniques and, when paired with high-resolution digital imaging, allow for large-scale profiling of gene expression at cellular resolution within a histological context. However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes an optimized, low-cost, small-footprint, high-throughput ISH procedure to detect gene expression patterns in 10-µm brain sections from zebra finches. It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium. The method is compatible with high-resolution digital imaging; it produces images with low background and a resolution approaching that of immunohistochemical methods. Approximately 180 slides can be processed each week using this protocol, but it can be scaled to accommodate a broad range of tissues from which cryosections can be obtained.
authors.affiliation: Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon 97239.
authors.forename: Julia B
authors.initials: JB
authors.lastname: Carleton
authors.affiliation: Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon 97239.
authors.forename: Peter V
authors.initials: PV
authors.lastname: Lovell
authors.affiliation: Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon 97239.
authors.forename: Anne
authors.initials: A
authors.lastname: McHugh
authors.affiliation: Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon 97239.
authors.forename: Tessa
authors.initials: T
authors.lastname: Marzulla
authors.affiliation: Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon 97239.
authors.forename: Katy L
authors.initials: KL
authors.lastname: Horback
authors.affiliation: Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon 97239.
authors.forename: Claudio V
authors.initials: CV
authors.lastname: Mello
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D003911:Dextrans / Q000737:chemistry; D046369:Finches / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D015347:RNA Probes / Q000378:metabolism
chemical_list: D003911:Dextrans; D015347:RNA Probes; C025614:sephadex