An optimized protocol for high-throughput in situ hybridization of zebra finch brain

Julia B. Carleton, Peter V. Lovell, Anne McHugh, Tessa Marzulla, Katy L. Horback, Claudio Mello

Research output: Contribution to journalArticle

11 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1249-1258
Number of pages10
JournalCold Spring Harbor Protocols
Volume2014
Issue number12
DOIs
Publication statusPublished - Dec 1 2014

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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