In situ Hybridization of Plant-parasitic Nematode Globodera pallida Juveniles to Detect Gene Expression
In situ Hybridization of Plant-parasitic Nematode Globodera pallida Juveniles to Detect Gene Expression
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DOI：
10.21769/bioprotoc.3372
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发表时间：
2019-09-20
期刊：
影响因子：
0.8
通讯作者：
Xiao, Fangming
中科院分区：
文献类型：
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作者：
Kud, Joanna;Solo, Nejra;Xiao, Fangming
在这项研究中，我们描述了一个标准的整体安装原位杂交方法，用于确定基因的时空表达模式，从Globodera spp。与更具侵入性的放射性标记方法不同，该技术基于安全、高度特异性的酶联免疫测定，其中通过与碱性磷酸酶（AP）缀合的抗DIG抗体（抗DIG-AP）检测与靶转录物杂交的地高辛（DIG）标记的反义探针。以5-溴-4-氯-3-吲哚基磷酸盐（BCIP）和硝基蓝氯化四氮唑（NBT）为底物，通过AP催化显色反应，观察到杂化分子。该方法可应用于自由生活的寄生前幼虫和胞囊线虫的早期内寄生阶段。
In this study, we describe a standard whole mount in situ hybridization method which is used to determine the spatial-temporal expression pattern of genes from Globodera 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-bromo4-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.