Name: long-term, high-resolution confocal time lapse imaging of arabidopsis cotyledon epidermis during germination
Uploaded: 2012-12-31T14:00:00
Description: Watch this Scientific Journal Video about Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination Video at JoVE.com

我们协助开发时间的推移协议和熊黛林Pillitteri建设的POLAR :: POLAR-EGFP感谢Rychel阿曼达。我们也感谢ABRC提供PM-RB建设的。这个协议是一个支持PRESTO奖来自日本的科学技术和代理的发展。极地研究还得到了大学，华盛顿版权研究基金（RRF，4098）和美国国家科学基金会（MCB-0855659）。 KMP是一个NSF研究所研究员（DGE-0718124），吉是HHMI的GBMF的研究者。

1。种子消毒<ol><li>准备种子消毒液：33％的家用漂白剂，0.1％的Triton X-100。 </li><li>广场种子携带所需的荧光记者的结构（S）和1.7 ml管型（S）和适用于1毫升灭菌水溶液。孵育15分钟nutator。 </li><li>在无菌罩，使用移液器删除解决方案，从管消毒，背后留下的种子。用1ml无菌水冲洗。重复四次。 </li><li>在4℃下孵育2天或以上。 </li></ol> 2。商会的媒体的制备<ol><li> 20毫升0.5％Bacto?...

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	<li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Embryonic+Cell+Lineage+of+the+Nematode+Caenorhabditis+elegans.">The Embryonic Cell Lineage of the Nematode Caenorhabditis elegans.</a> Developmental Biology. 100, 64 (1983).</li><li>Foe, V., Odell, G., Edgar, B. A., Bate, M., Martinez Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3+Mitosis+and+Morphogenesis:+Point+and+Counterpoint.">Chapter 3 Mitosis and Morphogenesis: Point and Counterpoint.</a> Development of Drosophila melanogaster. , (1993).</li><li>Vincent, C. A., Carpenter, R., Coen, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Lineage+Patterns+and+Homeotic+Gene+Activity+During+Antirrhinum+Flower+Development.">Cell Lineage Patterns and Homeotic Gene Activity During Antirrhinum Flower Development.</a> Current Biology. 5, 1449 (1995).</li><li>Beemster, G. T. S., De Vusser, K., De Tavernier, E., De Bock, K., Inz&#233;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Variation in Growth Rate between Arabidopsis Ecotypes Is Correlated with Cell Division and A-Type Cyclin-Dependent Kinase Activity. 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这个时间的推移激光共聚焦技术使荧光标记的蛋白质在拟南芥的子叶表皮，极性和动态变化的蛋白质的情况下，其功能是至关重要的一个正确的认识，单个细胞中的表达和定位的纵向研究。此前，一直持续时间延时成像用于检查拟南芥根真菌感染11和分生组织的生长5,12，但添加子叶表皮扩大这种技术的多功能性，并允许其使用额外的蛋白质，特别是协助气孔发展领域...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Bacto琼脂</td><td> BD </td><td> 214010 </td><td></td></tr><tr><td>一室幻灯片</td><td> Nunc公司（Thermo Scientific的） </td><td> 15536​​0 </td><td>或两个腔室（155379） </td></tr><tr><td>激光扫描共聚焦显微镜</td><td>蔡司</td><td> LSM700 </td><td>禅2009软件</td></tr><tr><td> 20倍物镜</td><td>蔡司</td><td> 420650-9901 </td><td> NA 0.8，规划复消色差透镜</td></tr><tr><td>解剖显微镜</td><td>奔驰（国家） </td><td> 431TBL </td><td>从下面点亮</td></tr><tr><td> ＃5镊子，生物学尖端</td><td> Roboz手术器械</td><td> RS-4978 </td><td>非常细的小秘诀是关键</td></tr></tbody></table>

long-term, high-resolution confocal time lapse imaging of arabidopsis cotyledon epidermis during germination

在整个发育序列体内的细胞行为的动态影像可以是一个强大的技术了解组织图案的机制。在动物发育，的关键细胞增殖和图案的事件发生得非常快。例如，在秀丽隐杆线虫的幼虫体内所有细胞分裂计划完成后6小时内受精，与七个有丝分裂周期1; 果蝇胚胎发育的16个或更多的有丝分裂发生在不到24小时2。与此相反，在植物发育的细胞分裂是缓慢的，通常在一天3,4,5的顺序。这会带来了独特的挑战和记录植物器官的细胞分裂和分化过程中的动态行为，需要进行长期实时成像调查信令，细胞命运和发展，在植物拟南芥表皮是一个很好的模型系统。。在子叶，这种组织是由空气和抗水的路面细胞穿插均匀分布的气孔，阀的打开和关闭控制的气体交换和水分损失。这些气孔的适当的间距，它们的功能是至关重要的，和它们的发展如下不对称分裂和细胞分化的步骤来产生有组织的表皮（ 图1）的序列。 该协议允许在表皮细胞和蛋白质的观察超过几天发展。这个时间框架的干细胞分裂和分化的表皮细胞，包括气孔和表皮路面细胞可以精确的文档。荧光蛋白可以稠合到感兴趣的蛋白的细胞分裂和分化过程期间，以评估它们的动态。这项技术使我们了解了一种新的蛋白质，POLAR 6的国产化，在气孔系细胞的增殖阶段，在日Ê的拟南芥子叶的表皮，它是在不对称分裂事件和细胞皮层不久之前分裂发生的一特征区移动到前面的细胞中表达。图片可以注册和流线型的视频轻松使用公共域软件的可视化动态和蛋白定位的细胞类型，因为它们随着时间的推移而改变。

<html>用这种方法收集的信息的时间点的一组是在图3中所示。细胞膜被标记为与RFP（PM-RB）和GFP融合POLAR蛋白质在其本地子（的POLAR :: POLAR-GFP）在30分钟的时间尺度，我们可以看到细胞分裂蛋白定位的变化他们前面。非对称细胞分裂在气孔血统形式的干细胞，如气孔，它保留接受进一步的不对称分裂的能力，meristemoids和他们的姐妹细胞的前体，称为气孔的血统地细胞（SLGCs）。的SL...

Watch this Scientific Journal Video about Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination Video at JoVE.com

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination Video (Video) | JoVE

我们描述了一个协议，用玻片和媒体的固定几天的发展，记录植物子叶共聚焦成像的表皮气孔分化。萤光标记的蛋白质，可以动态跟踪的表达和亚细胞定位，细胞分裂和细胞分化过程中增进了解其可能的作用。

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

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Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

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