We thank Ueli Grossniklaus (University of Z&#252;rich) for technical and financial support. We are thankful to Valeria Gagliardini, Christof Eichenberger, Arturo Bolanos and Peter Kopf for general lab support. This research was funded by the University of Z&#252;rich, grants from the Swiss National Foundation to CB (31003A_130722) and Ueli Grossniklaus (31003A_141245 and 31003AB-126006), and the Agence Nationale de la Recherche to DG (Programme ANR-BLANC-2012).

该过程在图1中的工作流描述，并设置为夹层和组织的嵌入在图2中给出。 1，组织固定<ol><li>收集20-30心皮中含有冰新鲜的BVO固定缓冲液离心管。 </li><li>修复组织30分钟轻微振荡在室温下。 </li><li>旋转含有固定剂的心皮中台式微量离心机1分钟，在400×g下的管中。 </li><li>小心地取出固定缓冲液和加入1 ml的PBT，把试管放在冰。 </li></ol> 2，解剖与嵌入<ol><li>准备5 Eppendorf管各200微升新鲜的，5％的丙烯酰胺组合。 </li><li>制备5的Superfrost载玻片预清洗，用70％乙醇及标记用铅笔。 </li><li>解冻20％的APS之一等份，每20％的国家行动方案，在冰上。 </li><li>取4-5心皮与切断端尖，地点他们在一个干净的玻片，去除多余的液体。 </li><li>使纵向切口用微细针取下心皮壁以释放列胚珠所示图2中 ，避免覆盖，用PBS（不超过10微升）的干燥。 </li><li>快速添加和混合12微升国家行动方案，12微升的APS与200微升丙烯酰胺混合等分。 </li><li>加入30微升的激活丙烯酰胺到解剖胚珠。 </li><li>盖在一个20×20毫米的盖玻片，让聚合在室温下，45-60分钟。 </li><li>用刀片去掉盖玻片。在此阶段，样品可以保持过夜，在4℃在含有PBS中的科普林氏缸。 </li></ol> 3，组织处理注：除3.2.1，3.2.3，3.3.2和3.4.3所有步骤都进行了科普林氏罐子，在室温下的化学罩80毫升解决方案。幻灯片被转移了一台尖镊子。 <ol><li>组织和澄清固定方式： <ol><li>孵育5分钟，在甲醇中。 </li><li>孵育5分钟，在乙醇中。 </li><li>孵育于乙醇30分钟：二甲苯（1:1）。 </li><li>孵育5分钟，在乙醇中。 </li><li>孵育5分钟，在甲醇中。 </li><li>孵育15分钟，在甲醇和PBT（1:1），补充有2.5％的甲醛。 </li><li>漂洗2×10分钟的PBT。在此阶段，幻灯片可以保持过夜，在4℃下</li></ol></li><li>细胞壁的消化： <ol><li>在冰上解冻的细胞壁消化混合物的等分试样。 </li><li>就拿从染色壶幻灯片，通过将它垂直于纸巾上沥干多余的液体。 </li><li>加入100μl的细胞壁消化混合比丙烯酰胺垫，盖上一个23 x 46毫米盖玻片。重复其他幻灯片。孵育2小时，在37℃的保湿室中（在材料中所述）。 </li><li>在PBT洗的幻灯片2×5分钟。 </li></ol></li><li> RNA酶A处理： <ol><li>就拿从染色壶幻灯片，沥干日Ë过量的液体和以前一样。 </li><li>孵育各滑动用100μlRNA酶A在100微克/毫升PBS中含1％Tween-20的1小时，在37℃下在潮湿的腔室中。 </li><li> 2×5分钟的PBT洗的幻灯片。 </li></ol></li><li>后固定和透： <ol><li>后固定20分钟的新鲜的PBT-F。 </li><li>冲洗的幻灯片中的PBT 10分钟。 </li><li> 2小时的PBS中透化，用2％Tween-20中在4℃下</li><li>冲洗的幻灯片2×5分钟的PBT。 </li></ol></li></ol> 4，免疫染色注意：对于这个步骤中，第一抗体的最佳浓度必须通过使用不同稀释度（1:200，1:500，1:1000）抗体进行检测。 <ol><li>孵育各滑动用100μl初级抗体在4℃下在PBS中稀释有0.2％Tween-20的12-24小时</li><li>在PBT为2-4小时，在下面轻轻摇动室温洗的幻灯片。 </li><li>应用在次级抗体1:200 PBS +0.2％Tween-20的24小时，在4℃下</li><li>在PBT中1小时以下轻轻摇动室温洗载玻片。 </li><li>染液用10μg/ ml的碘化丙锭的PBS中15分钟，然后冲洗在PBS中15分钟，在温和的晃动，在室温下进行。 </li><li>山中防褪色液封固补充10微克/毫升碘化丙啶。让我们通过激光共聚焦显微镜采集图像之前的安装介质硬化1小时。 </li></ol> 5。定量成像<ol><li>图像采集： <ol><li>利用激光共聚焦显微镜，最理想的是采用共振扫描模式，它可以更好地保存荧光信号在长时间成像23和63X甘油浸没透镜获得高清晰度的图像。 </li><li>测试采集参数，如激光强度，增益，针孔，像素大小和缩放因子在实验开始定义一个标准的收购程序严格遵循整个所有幻灯片的一致定量测量。 </li><li>验证的情况下的荧光染料之间的串扰。如果存在，建立一个顺序扫描。分开，而不是同时获得传输图像。 </li><li>与在x和y维度尽可能高的分辨率和在z维度（Nyquist的规则）2倍过采样进行串行，三维图像获取。 </li></ol></li><li>图像处理： <ol><li>使用商用或开源软件三维空间重建序列图像。 </li><li>定义轮廓的表面围绕在3D感兴趣的每个细胞核（或单元）。 </li><li>在每个信道量化荧光作为像素强度中的每个对象的总和。 </li><li>将数据导出到Excel进行统计分析。针对正常化，例如 DNA染色信号抗体信号。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

在开花植物中，女性生殖谱系是由几个细胞层，包括珠心和胚珠teguments包围，从而使细胞学染色整个安装技术上的挑战。在这里，我们提出了一个高效的协议使得大量适合细胞学染色，如免疫，DNA染色和荧光在整个贴装原位杂交胚珠的准备和处理。我们成功地使用它在拟南芥 21,22女性生殖生殖系的分析。该方法是高效的多张幻灯片可以并行进行不同的染色处理。它也?...

在开花植物，建立生殖谱系始于平滑肌细胞的分化，MMC女性和男性孢子母细胞。在MMC来自于胚珠原基的远端子珠心表皮细胞发育和小孢子母细胞从造孢组织发展中的花药药室，分别位于花器官1深处。校董会经过减数分裂，产生单倍体的孢子，然后产生在有丝分裂的配子体。雌配子体，或胚囊，由一个卵细胞，一个中央单元，两个助和三个antipodals的。雄配子体，或花粉，由一个营养细胞和两个精子细胞。而雄配子体仍然是一个相对平易近人对象 - 的研究中，雌配子体嵌入在胚珠内，自己包围在花心皮，从而造成分子和细胞学分析具体的挑战。然而，最近，激光辅助显微切割提供了一个优雅的解决方案，允许在MMC和雌性配子体细胞2-4转录组分析。除了 ​​候选基因表达分析， 例如使用RNA 原位杂交或报告基因检测，细胞学分析允许调查使用特定的直接细胞染色或间接免疫源性细胞成分的动态。特别是在使用FISH和DNA染色，与染色质修饰或染色质免疫成分一起细胞遗传学染色是核心的方法来阐明染色质动力学和核组织在拟南芥 5。通常情况下，减数分裂嗣继承已被很好地研究植物雄性meiocytes 6,7特定的染色体动态;进一步规模化，细胞特异性染色质重组，可能反映动态的表观遗传修饰已经花粉发育过程中8-10被描述。相比之下，由于到阴meiocyte和配子体的相对不便，这些调查保持在技术上难以适用，并且经常需要切片或手工剥离和酶消化（见下文）。此外，普遍缺乏在整个贴装光学清晰度是一个障碍生殖细胞中完整的胚珠高分辨率成像。 在整个安装胚珠一个经典的方法，染色体组织细胞学分析使用富尔根氏染色11-13。它涉及其导致蛋白质变性，从而使破坏的染色质结构的DNA的酸水解（使用次氯酸）。另外，在女性meiocytes和配子体细胞染色体的组织可以利用DAPI染色和免疫染色的半薄切片或解剖胚囊和MMC（例如见14-18）进行观察。但是很显然，人工剥离和切片可以是劳动密集和阻碍对大量染色质抗原表位的定性和定量分析。 在这里，我们提供了一个有效的协议准备了大量适合各种在整个贴装下游细胞学染色拟南芥胚珠。简言之，花芽孵育在固定剂溶液中，行胚珠从心皮解剖并嵌入在丙烯酰胺上滑动为已完成对花粉meiocytes 19,20。嵌入式胚珠进一步清除并固定在甲醇，乙醇，和细胞壁消化和透之前二甲苯。这些步骤可能的变化进行了探讨。样品随后可以用于DNA染色，免疫染色，以及鱼。编写模式是有效的，并允许并行实验装置（最多16个幻灯片可以在一天内进行制备为不同的下游分析）。所描述的治疗方法使信号均匀在全安装和保存完好的组织，细胞，和在生殖细胞和周围的珠心细胞，这些细胞有利于细胞类型之间的定性和定量的比较核的组织。校准，激光共聚焦显微镜为基础的高分辨率成像后的3维重建使荧光信号有意义的定量测量。我们成功地使用这个程序来分析染色质动态的区分MMC 21和发展中国家雌配子体22;我们在座的异染色质的分析，染色质免疫染色，免疫组化绿色荧光蛋白和FISH在整个安装胚珠的代表性结果。我们还认为，我们的协议将适用于其他植物组织和物种。

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an efficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in arabidopsis

在开花植物的体细胞到生殖细胞命运转变是在成人的植物花器官标志着孢子母细胞的规范（校董会）。女性SMC（大孢子母细胞，MMC）区分在胚珠原基和经历减数分裂。所选择的单倍体孢子然后经历有丝分裂，以形成多细胞雌配子体，这将产生配子，卵细胞和中央细胞，连同附件细胞。在MMC，meiocyte和雌配子体的胚珠内的有限的辅助功能是进行细胞学技术挑战性和细胞遗传学分析在单细胞水平上。具体地讲，蜂窝式或核抗原表位直接或间接的免疫检测是由植物细胞和单细胞成像内的试剂的渗透差的障是由缺乏在整个安装组织的光学透明性的批租。 因此，我们开发了分析原子核的有效方法耳组织和染色质修饰在整个安装嵌入式拟南芥胚珠高分辨率单细胞。它是基于解剖和固定胚珠嵌入在丙烯酰胺凝胶上的显微镜载玻片的薄层。嵌入式胚珠受到旨在提高组织的清晰度和通透的免疫染色试剂化学和酶处理。这些治疗维持细胞和染色质组织，DNA和蛋白质的表位。样本可以用于不同的下游细胞学分析，包括染色质免疫染色，荧光原位杂交（FISH），和DNA染色为异分析。共聚焦激光扫描显微镜（CLSM）成像，分辨率高，其次是三维重建允许在单细胞分辨率的定量测量。

我们提供了一个强大的协议大规模制备适合于在全安装细胞学染色拟南芥胚珠和处理。由于嵌入，胚珠保留的3维结构（ 图3）。此外，该组织处理，包括光学澄清，使成像的亚细胞结构的高分辨率。 图4显示了DNA染色在异染色质出现明亮，明确显眼灶（无反褶积用于此图片）整个安装胚珠原基。这些图像被用于分析在MMC和珠心（ 图4）21异的内?...

Watch this Scientific Journal Video about An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis at JoVE.com

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

我们在这里提供了一个高效，可靠的协议进行免疫染色，荧光原位杂交，DNA染色后在整个安装拟南芥胚珠定量，高分辨率成像。该方法已成功地用于分析染色质修饰和核架构。

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

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An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

13.1K 次观看.  University of Zürich. 我们在这里提供了一个高效，可靠的协议进行免疫染色，荧光原位杂交，DNA染色后在整个安装拟南芥胚珠定量，高分辨率成像。该方法已成功地用于分析染色质修饰和核架构。 