Protocol

Alzheimer's Disease and Frontotemporal Dementia

Volume 670 of the series Methods in Molecular Biology pp 207-230

Date:

Step-by-Step In Situ Hybridization Method for Localizing Gene Expression Changes in the Brain

  • Jorge J. PalopAffiliated withDepartment of Neurology, Gladstone Institute of Neurological Disease, University of California, San Francisco
  • , Erik D. RobersonAffiliated withDepartments of Neurology and Neurobiology, Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham
  • , Inma CobosAffiliated withNina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco

* Final gross prices may vary according to local VAT.

Get Access

Abstract

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.

Key words

In situ hybridization RNA RNA probes Riboprobes Brain sections Sliding microtome Cryostat EST Expressed sequence tag cDNA In vitro transcription Transcripts Digoxigenin-labeled probes NTB BCIP HNPP Fast red Light and fluorescence microscopy Alzheimer’s disease Frontotemporal dementia NPY ARC Nav1.1