In Situ Hybridization of Suprachiasmatic Nucleus Slices

The progress in the understanding of the molecular machinery of mammalian circadian clocks, in combination with the well-established role of the hypothalamic suprachiasmatic nucleus (SCN) as a master circadian clock, has provided an invaluable system for the study of the molecular basis of circadian rhythmicity. Using in situ hybridization (ISH) techniques that label specific clock-gene mRNAs within the SCN, researchers can now elucidate the core molecular oscillatory mechanisms underlying specific circadian physiological and behavioral phenotypes. In this chapter, two methods for ISH within the SCN are described. The first method is based on the fluorescent labeling of mRNA and is suitable for confocal microscopy analysis and double labeling techniques. The second method is based on the radioactive labeling of mRNA and is more sensitive and more adequate for the relative quantification of mRNA species.