An Optimized Protocol for High-Throughput In Situ Hybridization of Zebra Finch Brain

In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advances in non-radioactive methods based on chromogenic detection of digoxigenin (DIG)-labeled probes have increased spatial resolution compared to emulsion autoradiography, and when paired with high-resolution digital imaging have allowed for the large scale molecular profiling at cellular resolution within a histological context (e.g. Allen Brain Atlas, GenePaint.org; (Visel et al. 2004; Lein et al. 2007). However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes a low cost, small footprint, high-throughput ISH procedure for 10 μm sections developed to document brain gene expression in zebra finches (http://www.zebrafinchatlas.org). It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium (Replogle et al. 2008) and is based on previously described protocols for radiolabeled riboprobes (Clayton et al. 1988; Mello and Clayton 1994; Mello et al. 1997) that we have adapted for DIG-labeled (Lovell and Mello 2011; Lovell et al. 2013). Conditions have now been further optimized to produce cellular labeling approaching the resolution of immunohistochemical methods, low background, and compatibility with high-resolution digital imaging. This protocol allows a technician to process ~180 slides per week, and can be scaled to accommodate a broad range of tissues for which cryosections can be obtained.