In situ hybridization systems

Amarsham International Pic. (1994) A guide to radioactive non-radioactive in situ hybridization systems. 

Kishimoto J., Keveme E., Hardwick J. and Emson P. (1993). Localization of nitric oxide synthase in the mouse olfactory and vomeronasal system — a histochemical, immunological and in-situ hybridization study. Europ J Neurosci 5, 1684-1694. 

Abstract Quantitative receptor autoradiography methods have heen widely used over the last three decades to study the distribution and physiological role of a receptor in various tissues. This review provides an overview of in vivo and in vitro receptor autoradiography methods and their advantages as well as disadvantages in the study of receptors in the central nervous system. Comparison with immuno-histochemical and in situ hybridization methods is also highlighted in relation to the study of a given receptor in the nervous sytem. 

The complexities of protocols for fluorescent and chromogenic in situ hybridization necessarily entail careful attention to controls. In particular, the possibility of native enzyme activity or the presence of endogenous biotin in the experimental tissue should be considered, though this can be addressed by exposing control tissue to the detection system in the absence of probe. The relative merits of digoxigenin versus biotin, and some of the technical problems associated with each, have been previously discussed (Chevalier et al., 1997 Luo and jackson, 1999). 

Chawla MK, Lin G, Olson K, Vazdarjanova A, Burke SN, et al. 2004. 3D-catFISH a system for automated quantitative three-dimensional compartmental analysis of temporal gene transcription activity imaged by fluorescence in situ hybridization. J Neurosci Meth 139 13-24. 

In situ hybridization (ISH) permits the detection and iocaiization of DNA and RNA in a cytoiogicai preparation affixed to a microscope siide. Such detection and iocaiization is made possibie by hybridization of cellular DNA and/or RNA targets with nucleic acid probes tagged with a signal generating system such as a fluorochrome or enzyme. 

In the tissue sections, a hybridization is performed with a highly specific probe in order to detect the target (either amplified by indirect in situ RT-PCR or unamplified). For the preparation of nonradioactive digoxigenin-labeled probes, one should refer to the guidelines of the company manuals Genius M System User s guide for membrane hybridization and Nonradioactive in situ hybridization application manual (2nd ed., Boehringer Mannheim). 

Shughrue PJ, Lane MV, Merchenthaler I (1996) In situ hybridization analysis of the distribution of neurokinin-3 mRNA in the rat central nervous system. J Comp Neurol... 

Clark M, Massenburg GS, Weiss SRB, et al Analysis of the hippocampal GABA, receptor system in kindled rats by autoradiographic and in situ hybridization techniques contingent tolerance to carbamazepine. Brain Res Mol Brain Res 26 309-319, 1994... 

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