de la Iglesia Horacio O
Department of Biology, University of Washington, Seattle, USA.
Methods Mol Biol. 2007;362:513-31. doi: 10.1007/978-1-59745-257-1_41.
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.
对哺乳动物生物钟分子机制认识的进展，再加上下丘脑视交叉上核（SCN）作为主生物钟已确立的作用，为研究昼夜节律性的分子基础提供了一个极其宝贵的系统。利用原位杂交（ISH）技术标记SCN内特定的生物钟基因mRNA，研究人员现在能够阐明特定昼夜生理和行为表型背后的核心分子振荡机制。在本章中，将描述两种在SCN内进行ISH的方法。第一种方法基于mRNA的荧光标记，适用于共聚焦显微镜分析和双重标记技术。第二种方法基于mRNA的放射性标记，对mRNA种类的相对定量更敏感且更适用。
