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.

Figure 1

Improved protocol for high-throughput in situ hybridization on thin brain sections. (A–C) Examples of high signal, low background hybridizations demonstrating the specificity and cellularity of neuronal labeling in zebra finch cerebellum (A), song nucleus HVC (B), and the medial striatum (C). (D) Compared to control sections reacted without skim milk (panel D1), the addition of 1% skim milk to the TNB Blocking Buffer greatly reduces non-specific background labeling, particularly with long (> 3 day) chromogen incubations. (E) can further reduce non-specific background labeling. Non-specific labeling over the ventricle (E1; arrowheads) can be reduced by raising the temperature of the hybridization and post-hybridization washes from 65 to 67° C (E2). Abbreviations: gcl, granule cell layer; mcl, molecular cell label; pcl, Purkinje cell layer.