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Author:
Date:
2018-03-20
[Abstract] The spatio-temporal expression pattern of a gene provides important indications to better understand its biological function. In situ hybridization (ISH) uses a labeled complementary single-stranded RNA or DNA probe to localize gene transcripts in a whole organism, a whole organ or a section of tissue. We adapted the ISH technique to the plant parasite Meloidogyne spp. (root-knot nematode) to visualize RNAs both in free-living preparasitic juveniles and in parasitic stages settled in the plant tissues. We describe each step of the probe synthesis, digoxigenin (DIG) labeling, nematode extraction from plant tissue, and ISH procedure.
[摘要] 基因的时空表达模式为更好地理解其生物学功能提供了重要的指示。 原位杂交（ISH）使用标记的互补单链RNA或DNA探针来定位整个生物体，整个器官或一部分组织中的基因转录物。 我们将ISH技术应用于植物寄生虫
【背景】到目前为止，植物寄生性线虫的稳定转化尚未成功。 ISH能够在整个装载的Meloidogyne spp中分析体内时空基因表达。线虫。这些根结线虫在土壤中以微小蚓状幼虫（J2）形式孵化并感染宿主植物根部。 J2s穿透根部并迁移到根部维管柱状细胞。幼虫定居在根部，发育成J3和J4寄生幼鱼，诱导分化专化饲养细胞。线虫最终发育成梨形雌性，将在根表面释放数百个卵。在这里，我们报告了一个详细的协议来检测准备性整体安装J2s和寄生阶段中的单个RNA分子。寄生虫阶段的ISH需要在感染根部提取线虫前一天采取额外的程序。我们描述了在线虫整个组织中使用地高辛（DIG）标记的cDNA探针检测转录物。
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Author:
Date:
2014-04-05
[Abstract] The intestinal lamina propria contains a dense network of T cells, dendritic cells (DCs) and macrophages, which play an important role in local innate and adaptive immune responses. We have recently identified distinct subsets of DCs (Persson et al., 2013) and macrophages (Bain et al., 2013) in the human intestine. In addition, we have studied T cells in healthy and diseased intestine. Here, we describe two methods for isolating these cell populations: 1) enzymatic treatment and 2) migration based isolation. The enzymatic method can be used to isolate T cells, DC and macrophages, whereas the migration based ‘walk-out’ protocol is suitable for DC isolation, as these cells migrate out from the tissues.
[摘要] 肠固有层含有T细胞，树突细胞（DC）和巨噬细胞的密集网络，其在局部先天和适应性免疫应答中起重要作用。 我们最近在人肠中鉴定了不同的DC亚群（Persson等人，2013）和巨噬细胞亚群（Bain等人，2013）。 此外，我们研究了健康和患病肠中的T细胞。 在这里，我们描述了两种方法隔离这些细胞群体：1）酶处理和2）基于迁移的隔离。 酶法可用于分离T细胞，DC和巨噬细胞，而基于迁移的"走出"方案适合于DC分离，因为这些细胞从组织迁出。
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