Kud Joanna, Solo Nejra, Caplan Allan, Kuhl Joseph C, Dandurand Louise-Marie, Xiao Fangming
Department of Entomology, Plant Pathology and Nematology, University of Idaho, Moscow, ID, USA.
Department of Plant Sciences, University of Idaho, Moscow, ID, USA.
Bio Protoc. 2019 Sep 20;9(18):e3372. doi: 10.21769/BioProtoc.3372.
In this study, we describe a standard whole mount hybridization method which is used to determine the spatial-temporal expression pattern of genes from spp. Unlike more invasive radioactive labeling approaches, this technique is based on a safe, highly specific enzyme-linked immunoassay where a Digoxigenin (DIG)-tagged anti-sense probe hybridized to a target transcript is detected by anti-DIG antibodies conjugated with alkaline phosphatase enzyme (AP) (anti-DIG-AP). The hybrid molecules are visualized through an AP-catalyzed color reaction using as the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT). This method can be applied to both free-living pre-parasitic juveniles and early endoparasitic stages of cyst nematodes.
在本研究中，我们描述了一种标准的整体杂交方法，该方法用于确定[物种名称]基因的时空表达模式。与更具侵入性的放射性标记方法不同，该技术基于一种安全、高度特异的酶联免疫测定法，其中与靶转录本杂交的地高辛标记反义探针通过与碱性磷酸酶（AP）偶联的抗地高辛抗体（抗地高辛-AP）进行检测。杂交分子通过使用5-溴-4-氯-3-吲哚磷酸（BCIP）和氯化硝基四氮唑蓝（NBT）作为底物的AP催化显色反应进行可视化。该方法可应用于胞囊线虫的自由生活寄生前期幼虫和早期内寄生阶段。
