Fluorescence in situ hybridization on vibratome sections of plant tissues
Fluorescence in situ hybridization on vibratome sections of plant tissues
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DOI：
10.1038/nprot.2007.265
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Shaw, Peter
中科院分区：
文献类型：
--
作者：
Prieto, Pilar;Moore, Graham;Shaw, Peter
该协议描述了应用荧光原位杂交（FISH）的三维（3D）保存组织切片来自完整的植物结构，如根或小花。该方法是基于振动切片与共聚焦显微镜的组合。该协议提供了一个很好的工具，研究植物细胞核中的所有细胞类型的染色体组织，并已被用于单子叶植物和双子叶植物物种的组织。3D保存完好的组织的可视化意味着可以自信地识别细胞类型。例如，可以在减数分裂的所有阶段清楚地识别性母细胞，并且可以在其周围母体组织的背景下成像。FISH可用于定位着丝粒、端粒、重复区域以及独特区域，并且总基因组DNA可用作探针以可视化染色体或染色体片段。该方法适用于RNA FISH，并可与免疫荧光标记相结合。一旦所需的植物材料被切片，这取决于样品的数量，我们在这里提出的协议可以在3天内进行。
This protocol describes the application of fluorescence in situ hybridization (FISH) to three-dimensionally (3D) preserved tissue sections derived from intact plant structures such as roots or florets. The method is based on the combination of vibratome sectioning with confocal microscopy. The protocol provides an excellent tool to investigate chromosome organization in plant nuclei in all cell types and has been used on tissues of both monocot and dicot plant species. The visualization of 3D well-preserved tissues means that cell types can be confidently identified. For example, meiocytes can be clearly identified at all stages of meiosis and can be imaged in the context of their surrounding maternal tissue. FISH can be used to localize centromeres, telomeres, repetitive regions as well as unique regions, and total genomic DNAs can be used as probes to visualize chromosomes or chromosome segments. The method can be adapted to RNA FISH and can be combined with immunofluorescence labeling. Once the desired plant material is sectioned, which depends on the number of samples, the protocol that we present here can be carried out within 3 d.