ABSTRACT
Application of in situ hybridization has provided the opportunity to follow a functional measure of cellular activity within neurochemically defined populations of neurons over an interval sufficient to demonstrate rhythmicity. The most common probes used for in situ hybridization are oligodeoxynucleotides and “riboprobes”. To cut sections for hybridization, the brains are first removed from the –80°C freezer and allowed to equilibrate within the cryostat at a temperature range of –20 to –16°C. There are two approaches most often used for autoradiographic analysis of the hybridization signal, film autoradiography and emulsion autoradiography. For film autoradiography, the slides are placed in light-sealed autoradiographic cassettes and apposed to Kodak film until they are fully exposed. There are two sources of “nonspecific” labeling of tissue following in situ mRNA hybridization: cross-hybridization to related sequences and probe “binding” to non- RNA components in the tissue.
