Thisse Bernard, Thisse Christine
Department of Cell Biology, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA, 22903, USA.
Methods Mol Biol. 2014;1211:53-67. doi: 10.1007/978-1-4939-1459-3_5.
The in situ hybridization uses a labeled complementary RNA strand to localize a specific mRNA sequence in a tissue. This method is widely used to describe the spatial and temporal expression patterns of developmentally regulated genes. Here we describe a technique that employs in vitro synthesized RNA tagged with digoxigenin uridine-5'-triphosphate (UTP) to determine expression of genes on whole-mount zebrafish embryos and young larvae. Following hybridization, the localization of the specific transcript is visualized immunohistochemically using an anti-digoxigenin antibody conjugated to alkaline phosphatase that hydrolyzes the 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to 5-bromo-4-chloro-3-indole and inorganic phosphate. 5-Bromo-4-chloro-3-indole can be oxidized by nitro blue tetrazolium (NBT), which forms an insoluble dark blue diformazan precipitate after reduction.This protocol has been used for performing large-scale analyses of the spatial and temporal expression of the zebrafish genome, resulting in the description of more than 8,400 expression patterns that are available at the zebrafish information network (ZFIN.org) in the gene expression section.
原位杂交使用标记的互补RNA链在组织中定位特定的mRNA序列。该方法广泛用于描述发育调控基因的时空表达模式。在此，我们描述一种技术，该技术采用体外合成的、用洋地黄毒苷尿苷-5'-三磷酸（UTP）标记的RNA来确定斑马鱼全胚胎和幼体中基因的表达。杂交后，使用与碱性磷酸酶偶联的抗洋地黄毒苷抗体通过免疫组织化学法可视化特定转录本的定位，该碱性磷酸酶将5-溴-4-氯-3-吲哚磷酸（BCIP）水解为5-溴-4-氯-3-吲哚和无机磷酸。5-溴-4-氯-3-吲哚可被硝基蓝四唑（NBT）氧化，还原后形成不溶性深蓝色双甲臜沉淀。该方案已用于对斑马鱼基因组的时空表达进行大规模分析，从而描述了8400多种表达模式，这些模式可在斑马鱼信息网络（ZFIN.org）的基因表达部分获得。
