这项工作得到了苏黎世大学的支持, IEF. 居里夫人 (格兰特。TransEpigen-254797 对回历), 欧洲研究理事会的高级赠款 (授予 no。MEDEA-250358 到 U.G.), 和一个研究和技术开发项目 (赠款 MecanX U.G.) SystemsX.ch, 瑞士倡议在系统生物学。

1. 花和角果固定
<ol><li>从植物中收获的花朵和角果在第一朵花的开头同步。 注: 在这里使用的实验条件下, 植物开始开花大约21天后, 从 Murashige 和 Skoog (MS) 板块移植到土壤。种子被分层为3–4天在4°c 并且发芽或生长在 MS 板材在22°c/16 h 光和18°c/8 h 黑暗期间8到10天, 在被保留的盆栽下在营养丰富的土壤的幼苗在同样条件下 (图 1)。复制的数量取决于具体的研究目标, 但推荐每种治疗的至少5个花序 (从5个单独植物)。如果花是实验单位, 建议每复制至少10朵花。</li>
	<li>剪切花序或花 (图 1E-H) 使用小剪刀 (如, 指甲剪刀), 并立即放置在一个离心管 (为单花序/花固定) 或圆锥管 (为集体固定)含有新鲜制成的 Carnoy, 甲醇/乙酸, 或 FPA50 护 (取决于应用, 见下文) 在冰上。 注: 样品应完全浸入固定液中。</li>
	<li>将组织内的固定体保持在4小时至一夜4摄氏度。 注: 真空渗透可用于加快固定器的渗透到组织中, 但这可能会损害保存组织的结构。作者不实施真空渗透。</li>
	<li>取出固定剂, 添加足够的70% 乙醇, 以覆盖样品, 并将其返回到4摄氏度至少24小时;示例可以无限期地保留在此解决方案中。除去乙醇后, 立即进行下一步;要么解剖, 要么是 SDS 清理。</li>
</ol>2. 显微镜下的解剖
<ol><li>把新鲜的70% 乙醇放在一个手表制造商的玻璃上放置在一个小的培养皿支持。</li>
	<li>
将花序/花/角果放在这个刚制成的固定器上, 用镊子和注射器在显微镜下解剖。
	<ol>
<li>使用手表制造商的玻璃或类似的设备, 允许样品完全覆盖乙醇 (推荐) 的解剖的花序, 并为撕裂分离开花器官成熟的花朵 (萼片, 花瓣, 雄蕊可以解剖在这个阶段) 避免样品干燥的风险。</li>
	</ol>
</li>
	<li>
在幻灯片上解剖角果和小花蕾, 如下所述。
	<ol>
<li>将手表制造商的玻璃杯与预加工的样品放在一边, 并使用普通的幻灯片进行最后的解剖。</li>
		<li>将感兴趣的样品移动到幻灯片上, 并添加10µL 新鲜70% 乙醇。迅速在最后的解剖和添加10µL 乙醇, 如有必要, 以保持样品湿润, 不过量的液体。</li>
		<li>为从雄蕊释放花粉粒, 见步骤5.1。要解剖心皮和胚珠, 请按照图 2中描述的步骤操作。本程序适用于心皮形成发展的各个阶段, 包括绿色角果的发展阶段。 注: 如果有足够的乙醇结转, 请不要添加乙醇;在极小的液体中解剖非常小的样品要容易得多。在幻灯片上始终只保留感兴趣的器官 (萼片、花瓣、雄蕊、心皮、胚珠、种子或花粉粒)。该程序将确保幻灯片和盖玻片之间的均匀厚度, 对应于检查的器官的厚度, 导致更高的同质性, 从而提高显微检查的效率 (更高的目标标本数量在同一焦距平面上)。</li>
	</ol>
</li>
	<li>使用一小片吸纸来吸收和去除尽可能多的乙醇。快速进入下一步骤 (3、4或6节), 然后样品干燥。</li>
</ol>3. 以水合氯醛为基础的清洁和联合清除染色
注: 用 FPA50 固定材料获得水合氯醛净化效果最佳。
<ol><li>放置20µL 溶液 (水合氯醛/甘油, 改性霍耶的培养基, 先生的 4½, 或先生的 IKI-4½解决方案) 在试样轴承的幻灯片。使用一双注射器与传染性针, 根据需要定位标本, 减少他们之间的空间, 并翻转那些地方的利益机构面临向下。用针头除去剩余的气泡。</li>
	<li>将盖玻片侧放, 轻轻地将其放置, 轻轻地按压, 然后等待清除解决方案填补之间的空间。如果需要, 添加最小的清除解决方案, 以填充盖玻片下的整个空间。</li>
	<li>将幻灯片放在幻灯片持有者上, 并将其放在通风罩下。在至少4小时之后, 在接下来的第4-5 天进行显微观察。</li>
</ol>4. 联合亚历山大染色和4½的花药清除
注: 原始的亚历山大协议是基于释放花粉颗粒在幻灯片染色前。一种高效、更翔实、改良的亚历山大染色和清除程序是成熟而非开裂花药中成熟花粉粒的染色。
<ol><li>选择新收获的花蕾, 即将开放与非开裂的花药。 注: 不要服用较年轻的花蕾, 因为亚历山大染色是为了成熟的花粉粒, 不能有效地染色未成熟的花粉粒。推荐从不同的植物和花序收获的每治疗至少5朵花。</li>
	<li>准备一个96井板有足够的亚历山大的解决方案, 以淹没一个花蕾。通过去除萼片和花瓣来暴露花药, 并将它们浸入亚历山大的溶液中。把样品放在通风罩下 1–3 h。</li>
	<li>用4½溶液代替亚历山大的溶液, 将多井板放回通风罩下过夜孵化。</li>
	<li>使用镊子, 轻轻地将已清除的花蕾移到新的滑梯上。由于亚历山大的解决方案的一些结转可能会发生, 使用印迹纸尽可能地删除尽可能多的清除解决方案。</li>
	<li>解剖雄蕊并取出所有其他组织。按照3节中的步骤使用4½解决方案。</li>
</ol>5. 去除花粉颗粒中的花粉
注: 我们建议使用 Carnoy 的固定剂进行 DAPI 染色。
<ol><li>遵循1和2节所述的固定和解剖程序。到目前为止, 一个人应该有一个到许多雄蕊在幻灯片中的最低数量的70% 乙醇。</li>
	<li>使用印迹纸去除过量的乙醇, 并添加5–10µL DAPI 溶液。使用两个注射器的针头, 释放花粉颗粒内的少量溶液 (例如, 使用一个注射器固定雄蕊和另一个释放花粉粒)。如果溶液干燥, 再添加5µL 的 DAPI。</li>
	<li>去除雄蕊的所有碎片是一个关键的步骤, 只有花粉颗粒留在幻灯片和盖玻片之间。</li>
	<li>将盖玻片放在试样轴承的滑动上, 将其横向降低, 并添加填充空间所需的最小 DAPI 溶液量。把食指放在盖玻片上, 不挤压太多就能快速、牢固、短滑运动。 注: 为了测量滑动运动的必要力和振幅, 应多次练习这一步骤, 每次显微镜下检查结果。</li>
	<li>如果需要, 添加 DAPI 解决方案, 并将幻灯片放在一个潮湿的盒子在黑暗中的4°c 15 分钟到1小时. 在宽场荧光或共焦显微镜下观察样品在24小时内进行. 尝试将此过程与 SDS 清除相结合, 以检查是否进一步改善可以获得。</li>
</ol>6. 月桂酸钠 (SDS) 清除
注: 根据要分析的花器官, 以及研究人员的技能解剖非常柔软和小标本, SDS 治疗可以进行前 (为有经验的研究员) 或解剖步骤后 (为经验较少研究人员) 对甲醇-乙酸固定材料。
<ol><li>使用手表制造商的玻璃, 凹滑梯, 或离心管在室温下过夜, 在 1% SDS 和 0.2 N 氢氧化钠溶液中孵育解剖或非解剖样品。</li>
	<li>小心处理, 因为样品非常柔软, 外观透明。用清水冲洗几次。 注: 在添加染色溶液时, SDS 溶液的残余物可能导致沉淀,例如, 苯胺蓝。</li>
	<li>对于非解剖样品, 按照2节所述的解剖程序, 使用水代替70% 乙醇。解剖所需的组织后, 清除任何残留物和碎片, 以及任何多余的水, 用印迹纸, 然后添加20–40µL 染色溶液, 并进行步骤6.5。</li>
	<li>对于解剖样品, 小心地将样品从洗涤液中移出, 放到干净的滑梯上, 添加20–40µL 染色液。</li>
	<li>轻轻地将盖玻片放在幻灯片上。注意不要把准备工作压扁。如果组织太软, 请在幻灯片和盖玻片的拐角处放置任何薄分隔符(例如、磁带或破碎的盖玻片), 在幻灯片和盖玻片之间留下一个腔室状的空间, 这样盖玻片就不会粉碎试样。</li>
	<li>把所有的幻灯片放在潮湿的盒子里, 用铝箔盖住, 然后将它放在4摄氏度, 至少1小时. 在24小时内使用 widefield 荧光或共焦显微镜观察标本。</li>
</ol>

作者没有什么可透露的。

在整个花发育阶段, 在拟南芥的单个花序内存在许多花芽, 这为旨在表征治疗效果或发育特征的研究提供了独特的机会。同时跨越不同阶段的花卉发展。一个好参考点在不同的各自的植物之间是开头第一朵花的主要花序。植物被对待以这样方式开花是尽可能同步的 (例如, 3–4天分层在4°c 最大化同步种子发芽并且因而更加甚而开花), 并且只有在同一天打开第一朵花的植物是在处理和复?...

花卉是被子植物最重要的定义器官之一。开花植物出现了一些90–130百万年前的1, 和多样化如此之快, 他们迅速的外观被描述为一个 "可恶的神秘" 由查尔斯·达尔文2。植物研究人员在花卉开发中的兴趣是多种多样的。一些研究集中于了解花的进化起源, 或特定的解剖, 结构和功能性质的发展花卉3,4,5,6.花卉形态和结构的高度变异, 以及依赖于它们的性和无性繁殖模式, 使花卉具有高度复杂的结构。这导致了不同的努力描绘花卉器官的解剖和结构特征, 使用光和电子显微技术, 可以结合基因和分子调查7。此外, 作为水果和种子的来源, 花卉对人类和动物的营养至关重要。因此, 花卉和果实发育的特征对应用研究有许多影响, 包括在不断变化的环境下为日益增长的人类人口和生态保护战略提供粮食保障8,9,10。
在拟南芥中的花发育始于花诱导和营养分生组织向花序 (花) 分生的转化。花分化在花序分枝11的侧面侧向展开。花器官分化从外到花的中心逐渐形成同心轮生, 最终发育成萼片、花瓣、雄蕊和心皮7。这些花器官在不同的植物种类履行独特的营养, 保护和功能 (例如, 授粉吸引力) 角色, 与性器官保持男性和女性配子体发育的发展,分别12,13. 依次, 配子体发育分别区分了一对雄性 (精子) 和雌性配子 (卵和中央细胞), 它们结合在双重受精形成下一代, 受精卵和初生胚乳, 一个终端组织支持开发胚胎14,15。果实和种子的发育支持胚胎的生长、成熟和保存, 最终它的扩散。已经进行了广泛的研究, 以描述不同植物种类的花和胚胎发育, 特别是模型物种拟南芥7,16,17。
早期显微分析花卉开发是基于耗时的样品处理和观察技术, 如石蜡或树脂嵌入和切片, 结合光或电子显微镜。这些传统的显微技术通常与分子遗传学研究相结合, 如突变体的显微分析, RNA 的局部化通过原位杂交, 或蛋白质的免疫检测。最近在广域和共焦荧光显微学, 在先进的3维计算机辅助图像分析, 和不断细化的分子方法, 可用于微加工的全装标本, 已导致需要高效、最小的样品处理技术, 优先于定量分析。近年来, 对全芒动物标本的清洁技术的发展取得了长足的进展。它们通过使用水性尿素或糖基试剂 (例如、刻度、SeeDB、立方)18、19、20或选择性地去除脂质 (使用洗涤剂 SDS), 使样品透明在稳定水凝胶中嵌入样品;脂质的去除可以通过被动扩散 (例如、修改的清晰度协议21、协议-边缘-轮辋22) 或通过电泳 (原始清晰度协议23和动作的24) 来实现。在这种快速进步的鼓舞下, 一些衍生技术也正在涌现, 用于植物。
在本方法中, 本文重点介绍了模型拟南芥, 我们描述了正确解剖花芽、花朵和幼角果的程序, 并对各种染色和观察程序的全贴装样品进行了清理, 使用经典或最新的基于 SDS 的结算方法。给出了淀粉、胼胝和染色质染色的例子。虽然这些程序可能需要进一步的改进和适应, 当与其他物种使用, 我们希望他们将为进一步研究这些简单但关键的方法, 这是许多研究项目的出发点。

<table>
	<tbody>
		<tr>
			<td>Reagents and Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Scharlau</td>
			<td>ET00102500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Applichem</td>
			<td>A3686,2500</td>
			<td>100% Molecular biology grade</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Scharlau</td>
			<td>ME03062500</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde Solution</td>
			<td>Sigma-Aldrich</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Sigma-Aldrich</td>
			<td>81910-250 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>15307</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Roth</td>
			<td>3783.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>Fluka</td>
			<td>51198</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid</td>
			<td>Fluka</td>
			<td>69773</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma-Aldrich</td>
			<td>77607-250ML</td>
			<td>We used liquid phenol (use the density to find the required volume for your solution)</td>
		</tr>
		<tr>
			<td>Clove oil</td>
			<td>Sigma-Aldrich</td>
			<td>C8392-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Roth</td>
			<td>4436.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Fluka</td>
			<td>57665</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium iodide</td>
			<td>Merck</td>
			<td>5043</td>
			<td></td>
		</tr>
		<tr>
			<td>Malachite Green</td>
			<td>Fluka</td>
			<td>63160</td>
			<td></td>
		</tr>
		<tr>
			<td>Fuchsin acid</td>
			<td>Fluka</td>
			<td>84600</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Sigma</td>
			<td>7252</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>71690</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium di-Hydrogen Phosphate</td>
			<td>Applichem</td>
			<td>A1047,1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S9763-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>04347</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Applichem</td>
			<td>A2937,1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcofluor</td>
			<td>Sigma</td>
			<td>F6259</td>
			<td>Fluorescent brightener 28</td>
		</tr>
		<tr>
			<td>Auramine</td>
			<td>Chroma</td>
			<td>10120</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Aniline blue</td>
			<td>Merck</td>
			<td>1275</td>
			<td></td>
		</tr>
		<tr>
			<td>MS medium</td>
			<td>Carolina</td>
			<td>19-57030</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient-rich substrate</td>
			<td>Einheitserde</td>
			<td>ED73</td>
			<td></td>
		</tr>
		<tr>
			<td>Watch maker&#39;s glass</td>
			<td></td>
			<td></td>
			<td>No specific brand</td>
		</tr>
		<tr>
			<td>15 ml falcon centrifuge tubes</td>
			<td>VWR</td>
			<td>62406-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumond Forceps</td>
			<td>Actimed</td>
			<td>0208-5SPSF-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>DUMONT BIOLOGY</td>
			<td>0108-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD</td>
			<td>BD Plastipak 300013</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Preparation needle</td>
			<td>BD</td>
			<td>BD Microlance 304000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Scientific</td>
			<td>10143562CE</td>
			<td>cut edges</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Thermo Scientific</td>
			<td>DV40008</td>
			<td></td>
		</tr>
		<tr>
			<td>Humid box</td>
			<td></td>
			<td></td>
			<td>A plastic box with damp paper towel and slide supports inside</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fixatives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carnoy&#39;s (Farmer&#39;s) fixative</td>
			<td></td>
			<td></td>
			<td>Absolute ethanol : glacial acetic acid, 3:1 (ml:ml)</td>
		</tr>
		<tr>
			<td>Methanol/acetic acid fixative</td>
			<td></td>
			<td></td>
			<td>50 % (v/v) methanol, 10 % (v/v) glacial acetic acid in deionized water</td>
		</tr>
		<tr>
			<td>FPA50 fixative</td>
			<td></td>
			<td></td>
			<td>Formalin, propionic acid, 50% ethanol; 5:5:90 (ml:ml:ml)</td>
		</tr>
		<tr>
			<td>Clearing solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate/glycerol</td>
			<td></td>
			<td></td>
			<td>Chloral hydrate : glycerol : water, 8:1:2 (g:ml:ml). Can be used for all flower developmental stages and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td>
		</tr>
		<tr>
			<td>Modified Hoyer</td>
			<td></td>
			<td></td>
			<td>Gum arabic 7.5 g, chloral hydrate 100 g, glycerol 5 ml , water 30 ml. Can be used for all flower developmental stages and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td>
		</tr>
		<tr>
			<td>Herr&#39;s 4&#189; clearing fluid</td>
			<td></td>
			<td></td>
			<td>Lactic acid, chloral hydrate, phenol crystals, clove oil, xylene; 2:2:2:2:1, by weight. Can be used for all flower developmental stages (especially for stamen development) and for silique development with DIC microscopy. The best fixative is the formaline based FPA50</td>
		</tr>
		<tr>
			<td>SDS/NaOH solution</td>
			<td></td>
			<td></td>
			<td>Mix-dilute the the SDS and the NaOH stock solution to 1% SDS / 0.2 N NaOH (10x dilution). For all stages of flower and silique developmental stages. The best fixative is the methano/acetic acid fixative; the other two fixatives can also be used. Can be combined with calcofluor, auramine, DAPI, and aniline blue staining solution.</td>
		</tr>
		<tr>
			<td>SDS stock solution</td>
			<td></td>
			<td></td>
			<td>10 % (w/v) sodium dodecyl sulphate. Dissolve 10 g sodium dodecyl sulphate in 80 ml deionized water and make up to 100 ml with deionized water.</td>
		</tr>
		<tr>
			<td>NaOH stock solution</td>
			<td></td>
			<td></td>
			<td>2 N NaOH solution: dissolve 4 g of NaOH in 40 ml of deionized water and make up to 100 ml with deionized water</td>
		</tr>
		<tr>
			<td>Combined clearing and staining solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Herr&#39;s IKI-4&#189;</td>
			<td></td>
			<td></td>
			<td>To a standard 4&#189; (9 g in total) add: 100 mg iodine, 500 mg potassium iodide. This clearing solution can be used for all flower developmental stages and for silique development, either for increasing contrast or for characterizing starch dynamics. Use FPA50 for structural analysis and Carnoy&#39;s fixative for quantitative starch analysis.</td>
		</tr>
		<tr>
			<td>Alexander staining</td>
			<td></td>
			<td></td>
			<td>Ethanol 95% 10ml, malachite green (1% in 95% EtOH) 1 ml, fuchsin acid (1% in ddH2O) 5ml, orange G (1% in ddH2O) 0.5ml, phenol 5g, chloral hydrate 5g, glacial acetic acid 2ml, glycerol 25ml . This clearing/staining alone or in combination with Herr&#39;s 4&#189; solution can be used to evaluate pollen abortion in flowers with mature and tricellular pollen grains. It&#39;s used on freshly harved non-fixed material.</td>
		</tr>
		<tr>
			<td>Staining solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcofluor solution</td>
			<td></td>
			<td></td>
			<td>Calcofluor 0.007% in water (g:ml). Originally used as an optical brightner. Can be used for staining cellulose, carboxylated polysaccharides and callose in cell walls. Frequently used to stain the intine of the pollen grain. All three fixatives can be used with this solution.</td>
		</tr>
		<tr>
			<td>Auramine solution</td>
			<td></td>
			<td></td>
			<td>Auramine 0.01% in water (g:ml). This lipophilic fluorscent dye can be used for staining cuticles, cutin, and exine among others. All three fixatives can be used with this solution</td>
		</tr>
		<tr>
			<td>Calcofluor-Auramine mixture</td>
			<td></td>
			<td></td>
			<td>Auramine solution : Calcofluor solution, 3:1. Can be used for a combined staining by both solutions. Other proportions can be assayed maintaining a smaller proportion of calcofluor with respect to auramine.</td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td></td>
			<td></td>
			<td>DAPI 0.4 ug/ml, 0.1 M sodium phosphate buffer (pH 7), 0.1% Triton-X-100, 1 mM EDTA. This solution can be used for staining chromosome spreads during male and female meiosis, and cell nuclei of any tissue. Frequently used for studying pollen grain development. Carnoy&#39;s and methanol/ acetic acid are the best fixatives for this solution. Formaldehyde-based fixatives such as FPA50 may interfere with the staining. Excitation in the UV and maximum emission around 461 nm.</td>
		</tr>
		<tr>
			<td>Sodium phosphate buffer (0.1 M)</td>
			<td></td>
			<td></td>
			<td>Proton receptor: 0.2 M Na2HPO4, proton donor: 0.2 M NaH2PO4, ratio proton donor / proton receptor: 1.364 ( for a pH 7)</td>
		</tr>
		<tr>
			<td>Aniline blue solution</td>
			<td></td>
			<td></td>
			<td>0.1% (w/v) aniline blue, 108 mM K3PO4 (pH 11), 2% glycerol. This solution can be used for staining callose and cellulose of many stages of development (e.g callose deposition in male and female terads, callose plugs in pollen tubes). Excitation in the UV and maximum emission around 455. It can also be excited at 514 nm with emission in the red for cell content staining.</td>
		</tr>
	</tbody>
</table>

whole-mount clearing and staining of arabidopsis flower organs and siliques

由于其强大的分子遗传学研究工具,拟南芥是植物生物学中最突出的模型物种之一, 尤其是植物繁殖生物学。然而, 植物形态学, 解剖和超微结构分析传统上涉及耗时的嵌入和切片程序, 明亮的领域, 扫描和电子显微镜。最近在共焦荧光显微术, 先进的3维计算机辅助显微分析和不断细化的分子技术, 用于微加工的全装标本, 已导致增加的需求开发高效、最小的样品处理技术。在本议定书中, 我们描述了正确解剖拟南芥花和角果的技术, 基本的清除技术, 以及对生殖结构进行整体观测的一些染色程序。

拟南芥属于 Brassicacea 家族, 具有伞房花序(图 1) 中排列的两性花的花序。每朵花有四萼片, 四个花瓣, 六雄蕊 (四长和两个短), 和一个合心皮雌蕊由两个先天融合的心皮 (图1F-H) 安排在四同心螺纹 25, 26.拟南芥有 tenuinucellate, 胚珠胚珠排列在顶叶胎的两行 (每行?...

Watch this Scientific Journal Video about Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques at JoVE.com

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

拟南芥花器官和角果的全装清和染色

在本议定书中, 我们描述了适当解剖拟南芥花和角果的技术, 一些基本的清洁技术, 以及对生殖结构的全装观察的染色程序。

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

whole-mount-clearing-staining-arabidopsis-flower-organs

Research

JoVE Journal

Developmental Biology

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

16.6K 次观看.  University of Zurich. 在本议定书中, 我们描述了适当解剖拟南芥花和角果的技术, 一些基本的清洁技术, 以及对生殖结构的全装观察的染色程序。

视频: Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

器官培养和鼠标沃尔弗导管的全山免疫染色

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

科研

发育生物学

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

协议的可视化类固醇机关及其互动器官在果蝇免疫染色<em&gt;果蝇</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

成人耳皮肤全安装共焦显微术: 一种研究神经血管分支形态发生和免疫细胞分布的模型系统

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

三维脊髓前庭和听觉感官器官的培养

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...