Step-by-step in situ hybridization method for localizing gene expression changes in the brain.

RNA in situ hybridization is a powerful technique for examining gene expression in specific cell populations. This method is particularly useful in the central nervous system with its high cellular diversity and dynamic gene expression regulation associated with development, plasticity, neuronal activity, aging, and disease. Standard quantitative techniques such as Western blotting and real-time PCR allow the detection of altered gene or protein expression but provide no information about their cellular source or possible alterations in expression patterns. Here, we describe a step-by-step RNA in situ hybridization method on adult and embryonic brain sections for quantitative neuroscience. We include fully detailed protocols for RNase-free material preparation, perfusion, fixation, sectioning, selection of expressed sequence tag cDNA clones, linearization of cDNA, synthesis of digoxigenin-labeled RNA probes (riboprobes), in situ hybridization on floating and mounted sections, nonradioactive immunohistochemical detection of riboprobes for light and fluorescence microscopy, and double labeling. We also include useful information about quality-control steps, key online sites, commercially available products, stock solutions, and storage. Finally, we provide examples of the utility of this approach in understanding the neuropathogenesis of Alzheimer's disease. With virtually all genomic coding sequences cloned or being cloned into cDNA plasmids, this technique has become highly accessible to explore gene expression profiles at the cellular and brain region level.