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

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

Author information

1
Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, Oregon 97239.

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.

PMID:
25342071
PMCID:
PMC4589143
DOI:
10.1101/pdb.prot084582
[Indexed for MEDLINE]
Free PMC Article

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