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

Cold Spring Harb Protoc. 2014 Oct 23;2014(12):1249-58. doi: 10.1101/pdb.prot084582.

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.

MeSH terms

  • Animals
  • Brain / cytology
  • Brain / metabolism*
  • Dextrans / chemistry
  • Finches / metabolism*
  • High-Throughput Screening Assays / methods*
  • Immunohistochemistry
  • In Situ Hybridization / methods*
  • RNA Probes / metabolism

Substances

  • Dextrans
  • RNA Probes
  • sephadex