这项工作由 NIH/aha R56 HL128646 和 Mindich 儿童健康和发展研究所 (MCHDI) 在 ISMMS (北达科他州) 资助。比由 NIH/NIDCR 培训 T32 HD075735 支持。显微镜和图像分析是在位于西奈山的伊坎医学院的显微镜中心进行的, 这部分由西奈山 P30 CA196521 悌癌症研究所支持。

此处描述的所有方法都已由西奈山医学院的伊坎医学院的机构动物保育和使用委员会批准. 
 1. 收集和处理心脏新月期胚胎 <ol> <li> 交配一只肥沃的雌性老鼠。
	<li> 检查是否存在阴道交配插头使用钝探针或镊子。当天中午的插头检测被认为是胚胎日 (E) 0.5 (见 图 1A 完整的时间线). 
	注: 插头应在上午检查, 因为他们是在整个一天丢失. </li>
	<li> 在 8 日 (E8.25) 的早晨, 通过 CO 2 吸入, 或根据当地和机构的规定牺牲怀孕的大坝. 
	注意: 精确的时间可以是应变依赖, 并应由形态学经验. </li>
	<li> 用70% 乙醇喷洒鼠标的腹部, 清洁区域, 减少脱落。通过腹部切开皮肤和身体壁来揭露内脏. </li>
	<li> 定位子宫并小心地从动物身上取出整个子宫角。首先从一个输卵管切开, 修剪脂肪远离子宫, 同时进行宫颈末端。通过宫颈切开并继续到另一端输卵管的上端, 切开整个子宫. </li>
	<li> 将子宫放在10厘米的盘中, 用磷酸盐缓冲盐水 (PBS, pH 值 7.4) 冲洗掉多余的血液。通过切割 mesometrium 之间的 deciduum, 其中包含了胚胎, 解剖子宫。用新鲜的 PBS 将胚胎移植到一个6厘米的盘子里. </li>
	<li> 在解剖显微镜下, 用细钳 (#5) 将子宫组织移离蜕组织. </li>
	<li> 使用镊子, 仔细切片的 deciduum 的胚胎一半的尖端, 以揭示胚胎。捏 deciduum, 把胚胎取出, 小心地把胚胎取出. </li>
	<li> 尽可能多地解剖外组织而不损害胚胎的形态学 ( 图 1B ). </li>
	<li> 使用转移吸管将胚胎放置在1.5 毫升的试管中, 并在冰上放置新鲜的 PBS 和地方。对其余的胚胎重复 1.7-1.9, 然后继续. </li>
	<li> 吸 pbs, 用 pbs 冲洗一次。吸气 PBS. 
	注: 尽量去除尽可能多的 PBS, 而不损害或干燥的胚胎。建议在所有步骤中手动删除解决方案, 以避免丢失胚胎. </li>
	<li> 在 RT 中为1小时在 PBS 中修复4% 甲醛 (粉煤灰) 的胚胎. 
	注: 胚胎可在4和 #176 一夜之间固定 (O/N); C. </li>
	<li> 用 PBS 冲洗三次。将胚胎保持在4和 #176; C 直到准备好进行免疫荧光. 
	注意: 暂停点。胚胎可以安全地储存在 PBS 4 和 #176; C 几个星期. </li>
</ol> 2。免疫荧光染色 注意: 下面的孵化条件可以调整以适应不同的时间安排。建议使用轻柔摇动或摇动所有长潜伏期的步骤. 
<ol> <li> 删除 pbs, 并在 pbs 中添加1毫升的阻断缓冲器 (0.5% 皂苷、1% 牛血清白蛋白 (BSA)). </li>
	<li> RT 时至少孵育4小时。这也可以在4和 #176 中完成; C. </li>
	<li> 删除阻塞缓冲区, 并添加在阻塞缓冲区中稀释的主抗体混合物。在4和 #176 孵育 O/N; C. 
	注: 请参阅材料部分以描述所使用的抗体和建议的稀释。抗体稀释应根据经验确定。推荐使用 Nkx2-5 作为心脏新月的参考色斑, 是下游图像分割和分析步骤的关键. </li>
	<li> 通过吸入去除主抗体. </li>
	<li> 在 PBS 中每1小时清洗3次, 每次使用0.1% 个海卫一. </li>
	<li> 去除洗涤和添加二次抗体混合物稀释在堵塞缓冲。在 RT 处孵育3小时。这也可以在4和 #176 中完成; C. </li>
	<li> 在 PBS 中用0.1% 个海卫一清洗3次, 每次1小时。这也可以在4和 #176 中完成; C. </li>
	<li> Counterstain 与4和 #39;, 6-diamidino-2-吲 (DAPI) 在 PBS 中为 10 min. 
	注: 此 counterstain 可同时进行二次抗体. </li>
	<li> 在 PBS 中用0.1% 个海卫一清洗2次, 每次5分钟. </li>
	<li> 缓慢地暂停防安装介质中的胚胎 (2% 伏无食子 nPG), 90% 甘油, 1x PBS;有关详细信息, 请参阅参考资料部分)。安装前允许平衡至少1小时。轻轻地弹管, 帮助胚胎落入防溶液. 
	注意: 暂停点。胚胎可以储存数天, 在防的解决方案, 直到准备安装和图像. </li>
</ol> 3。显微镜下安装胚胎 <ol> <li> 使用胶布磁带或硅胶垫片准备用于安装的显微镜幻灯片。如果使用胶布的磁带, 使两个平行的堆栈5-6 层约15-20 毫米分开。这将留下足够的空间来放置胚胎并确保片 ( 图 1C ). </li>
	<li> 放置15和 #181; 在胶布磁带之间的幻灯片上放置防。小心地将一个胚胎转移到滑梯上. 
	注意: 一个以上的胚胎可以放在一个幻灯片上。然而, 随后的方向步骤是更困难的多胚胎, 和长的成像时间可能导致光漂白. </li>
	<li> 在解剖显微镜下使用细钳, 定位胚胎, 使前侧面远离幻灯片与身体轴与幻灯片的长轴线。有关安装安装的示意图, 请参见 图 1C . </li>
	<li> 小心地将一个片放在样品上, 将一侧放在一叠胶布胶带上, 然后用镊子轻轻地降低片, 直到它与另一条带子接触. 
	注意: 步骤3.3 和3.4 是确保胚胎成像正确方向的关键。通过降低片沿后向前方的方向, 胚胎不应该滚动和心脏新月区将保持远离幻灯片, 这是理想的倒置显微镜成像. </li>
	<li> 使用吸管在幻灯片和片之间添加额外的防, 以防止样品在存储/成像过程中消失. </li>
</ol> 4。共焦成像 <ol> <li> 使用最高放大率目标获取图像, 使整个心脏新月区在一个视场中被捕获。使用奈奎斯特采样率来确定 x-y-z 像素尺寸。大多数显微镜制造商将有一个和 #34; 优化和 #34; 按钮以确保奈奎斯特取样. 
	注: Nkx2-5 染色在这里是有益的, 因为它将描绘整个心脏新月区。较小的 Z 步尺寸 (采样) 将产生更好的3D 建模结果, 但可能会导致延长采集时间和/或漂白一些样品. </li>
	<li> 使用标准 best-imaging 实践设置成像参数。即, 使用尽可能低的激光强度, 以获得良好的信噪比为每个荧光, 并选择增益和抵消水平, 产生一个广泛的动态范围不饱和的信号. 
	注: 与将直接比较的样品之间的成像参数保持一致。这对于下游3D 分析和量化尤为重要. </li>
</ol> 5。图像分析和定量3D 建模 注意:对于该协议, 使用 Imaris 软件包对图像进行了分析。类似的分析可能使用其他的软件包。下面的说明介绍了一个引用通道 ( 即 Nkx2-5) 和一个实验通道 ( 即 YFP) 的分析管道。可以通过重复这些步骤来分析其他通道。提供了一个示例数据集, 可用于复制下面的分析. 
<ol> <li> 将原始图像数据集加载到 Imaris 竞技场视图中, 并在超过视图中为所需的胚胎打开文件。数据应在3D 视图中加载 MIP (最大) 模式。打开 "显示调整" 窗口, 并只选择参考通道. </li>
	<li> 如果需要, 可调整图像的阈值, 以便更好地进行可视化。对于这个分析, 如果信噪比过低, 则调整伽玛. 
	注意: 如果执行伽玛校正, 这是非线性调整, 调整后的图像不能用于强度测量或比较。在这个阶段, 你可以进行进一步的图像预处理 (如光照校正, 高斯模糊或其他过滤), 如果你的信噪比是不满意的。如果选择进行预处理, 则必须对数据集中的所有图像执行相同的预处理步骤. </li>
	<li> 使用曲面算法对话框为参考通道创建新曲面. 
	<ol> <li> 选择和 #34; 只对感兴趣的区域和 #34 进行细分; 复选框. </li>
		<li> 调整边界框区域, 使其包含感兴趣的区域 (ROI), 即 "参考色斑" 所描述的 心脏新月区). </li>
		<li> 从源通道下拉菜单中选择参考通道。默认的平滑设置 (等于 z 像素大小) 通常是足够的, 但可能需要经验优化。对于此分析, 使用平滑等于 z voxel-size 的一半. </li>
		<li> 按绝对强度进行阈值化。要确保曲面不延伸到通道信号之外, 请使用滑块调整下限. 
		注意: 对于某些可以很容易划定细胞的图像, 可以包括 #34; 分割触摸的对象和 #34; 算法, 以便进一步将信号与背景分开. </li>
		<li> 按体素数字或体积筛选对象, 并拒绝背景 (小或在 Nkx2-5 区域之外的区域中找到) 该算法可能生成的对象。完成算法. </li>
	</ol> </li> <li> 使用新创建的曲面, 为实验通道创建一个屏蔽通道. 
	<ol> <li> 如果选定了曲面, 则从 "编辑" 窗口中选择 #34; 掩码 Sel... #863; #34; 函数. </li>
		<li> 选择感兴趣的实验通道 ( 即 YFP)。在应用掩码和 #34 之前确保 #34;D 重复通道; 选中复选框. </li>
	</ol> </li> <li> 在显示调整窗口中只选择屏蔽通道。按照步骤 5.3-5.3. 5, 为掩码通道创建一个新曲面. 
	注意: 此时调整每个掩码的外观有助于3D 模型的可视化: 选择每个曲面并选择 "颜色" 选项卡. 根据需要调整基本颜色和透明度。重叠时, 曲面颜色不合并. </li>
	<li> 若要获取所需的体积数据, 请选择 "统计" 选项卡。在和 #34;D etailed 和 #34; 子, 选择和 #34; 平均值和 #34; 从下拉菜单中。在生成的表的 Sum 列中可以找到该曲面的总体积. 
	注意: 如果生成的曲面包含错误的片段 ( 即 片段, 这些片断是在分割期间未排除的背景信号或从主表面间断的区域), 则可能需要手动计算总卷, 或按体的体积或数量过滤曲面, 并确保计算中只包含信号段。通过选择和 #34, 所有的值和 #34; 从下拉菜单中, 可以找到每个曲面部分的音量。选择每个值将使相应区域显示为黄色, 这有助于确定要排除的值. </li>
</ol>

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H., Mohn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryonic+explant+culture+system+for+analysis+of+hematopoietic+and+vascular+development.">Mouse embryonic explant culture system for analysis of hematopoietic and vascular development.</a> Methods Mol. Med. 105, 231-256 (2005).</li></ol>

作者没有什么可透露的。

上面的协议描述了从高质量的 post-implantation 小鼠胚胎免疫荧光图像中获取定量数据的策略。当正确执行时, 通过这些步骤生成的3D 体积数据可用于胚胎内多个域的比较和交叉分析。表面信号掩蔽方法的描述是特别使用时, 研究新的细胞数量与公认的参考结构比较。
我们认为, 这种方法比现有方法具有明显的优势。全贴装免疫荧光与广域成像可以提供组织级信息, 但缺乏细胞分辨率。反之, 序列切片的免疫荧光可以给出详细的细胞级分辨率, 尽管这些数据缺乏整个贴装成像的三维优势。虽然三维共焦成像解决了这些缺点, 但很少有用户利用这些数据的数量潜力, 我们希望我们的协议将允许更多的研究人员充分利用他们收集的数据。
在这里, 这一方法已被证明使用标记 Nkx2-5, 以描绘心脏新月和检查存在的 Foxa2Cre:YFP 血统跟踪细胞在该领域。但是, 由于能够同时映像至少三血统和生成的高分辨率三维数据, 因此该协议可能对许多领域的研究人员都是有用的。我们建议, 这种方法可以适用于多个胚胎阶段, 器官系统和血统, 通过对协议的小调整, 根据该系统的具体需要。根据我们的经验, 这项协议是直接适用于一系列的胚胎年龄 (E6.5-E9.5 在最低限度) 和有限的适应。19我们没有遇到与所列潜伏期相同的胚胎的通透性或抗体穿透的问题, 尽管我们预计, 如果有较厚或密度更大的组织, 这些 E9.5 将不得不拉长, 以达到完全穿透。
这里描述的分析是使用专有的软件包进行的, 但是可以被改编为在针对大数据处理而优化的替代专有软件包中执行。或者, 类似的管道可以使用计算语言或开源软件包轻松地实现, 这将导致可共享协议数据库的快速开发。全球科学家网络的发展, 可以获得免费获得的用于图像采集和分析的工具和协议, 将迅速推进该领域并增加研究的重复性。17
根据我们的经验, 对这种方法的主要限制是与成像深度有关, 特别是在后期的胚胎阶段, 并将很大程度上取决于所使用的显微镜设置的细节。同样, 如何为映像安装示例将取决于用户的特定需要。我们发现, 把最接近透镜的区域放在提高成像深度和降低背景信号, 我们建议使用分层双面胶带安装, 因为它使用户能够创建一个会议厅, 符合他们的特定需要。使用 glycerol-based 安装介质与防试剂, 和均衡样品在这个媒体前安装, 强烈建议抑制漂白。最后, 本分析所使用的样本几乎是透明的;用户可能需要为更高级的胚胎阶段、组织碎片或 organoids 合并清除步骤, 并且确定给定应用程序的最佳结算协议将需要进行经验测试。
在心脏形态发生的情况下, 可以使用各种成像方法。在这里使用共焦显微镜, light-sheet 显微镜非常适合这些研究, 因为获得的图像的速度, 分辨率和信噪比。20我们预计这一新技术的可用性将进一步提高类似成像方法的实用性和丰富性。同样, 连续推进荧光记者,21团, 染料,22和活胚培养23,24将导致这些研究移动到四维度, 添加时间的元素, 并关于胚胎结构在发育过程中如何形成的更多信息。

器官发生的研究一直依赖于发育中胚胎的形态发生事件的观察。这些研究往往依赖于使用荧光染料或血统追踪记者与标签的定义参考人口。1通过比较这些标签的相对位置, 可以收集有关某一群体的起源、移动或最终贡献的信息。移植和命运映射实验使用形态学地标或染料射入 non-motile 血统定义兴趣细胞的出发点, 然后被审查为贡献对被开发的胚胎。2,3,4,5遗传谱系跟踪实验使用与定义明确的报告等位基因相同的概念, 用于标记细胞数量, 而无需进行实验操作。这些方法的关键是能够确定, 具有高空间分辨率, 实验和参考标签的位置。这些方法在前发育和外植体研究方面取得了显著进展。6,7,8,9
近年来, 心脏形态发生的发展事件越来越受到人们的广泛描述。10这一研究领域的主要发现之一是描述了一些可以通过独特标记的表达来区分的祖群。11这些种群包括第一和第二个心脏领域 (FHF 和 SHF), 它们存在于胚胎前侧的心脏新月中 (E) 8.25 的小鼠发育。12这些种群经常通过广泛的显微镜的结合进行检查, 这提供了组织级的信息, 并通过免疫荧光分析进行了序列切片, 这提供了高的细胞分辨率, 但two-dimensional 空间信息。13因此, 虽然这些研究大大提高了我们对心脏发育的认识, 但现有的方法在这些阶段对形态发生的深入定量分析有限, 因此需要采取方法来检查这些人口的组织在一个整体有机体水平。
最近的进展, 共焦显微镜和3D 图像分析允许高分辨率和高通量算法重建的细胞和结构原位相对容易, 从而铺平了道路的详细研究复杂细胞结构。14随着计算能力的提高和大数据管理算法的发展, 既需要处理成像数据集大小的指数增长, 现在可以完全自动化分析。15对成像数据集的自动化分析具有无偏见的优点, 但它只与输入数据集的质量一样可靠;因此, 必须在采集和图像预处理过程中使用最佳, 以确保最高质量、无偏见的分析。16协议可以完全自动化并共享以实现重复性, 而专有软件所使用的算法也可通过库来方便地使用, 这些用户熟悉现代专有或开源开发工具。17
下面的协议解释了在心脏发育过程中对一个明确的器官形成模型进行分析的必要步骤。具体来说, 该协议描述了如何 (1) 收获和解剖心脏新月期胚胎, (2) 执行全贴装免疫荧光的参考 (Nkx2-5) 和实验 (Foxa2Cre:YFP18,19) 标记, (3)使用共聚焦显微镜制备和成像胚胎, 最后 (4) 使用先进的三维方法对结果图像进行分析和量化。在这里, 以心脏新月为例, 通过适当的修改, 本协议可用于分析肠早期节期胚胎的多血统。

<table>
	<tbody>
		<tr>
			<td>Blunt probe</td>
			<td>Roboz</td>
			<td>RS-9580</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS-8100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Roboz</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Roboz</td>
			<td>RS-5912</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma Aldrich</td>
			<td>84510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A8022</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>RPI</td>
			<td>A4490</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Nkx2-5</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-8697</td>
			<td>Used at 1:100-1:500</td>
		</tr>
		<tr>
			<td>Chicken anti-GFP</td>
			<td>abcam</td>
			<td>ab13970</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Islet1</td>
			<td>abcam</td>
			<td>ab109517</td>
			<td>Used at 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti-Hcn4</td>
			<td>Millipore</td>
			<td>AB5808</td>
			<td>Used at 1:100</td>
		</tr>
		<tr>
			<td>488 anti-chicken</td>
			<td>Jackson Immunoresearch</td>
			<td>703-546-155</td>
			<td>Reconstituted in water and stored at -20 &#176;C in final concentration of 50% glycerol. Used at 1:500.</td>
		</tr>
		<tr>
			<td>555 anti-goat</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21432</td>
			<td>Used at 1:500</td>
		</tr>
		<tr>
			<td>647 anti-rabbit</td>
			<td>Jackson Immunoresearch</td>
			<td>711-606-152</td>
			<td>Reconstituted in water and stored at -20 &#176;C in final concentration of 50% glycerol. Used at 1:500.</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Propyl gallate</td>
			<td>Sigma Aldrich</td>
			<td>2370</td>
			<td>Stock solution is 20% w/v in DMSO. Working solution prepared by mixing 1 part 10x PBS with 9 parts 100% glycerol and slowly adding 0.1 part stock solution.</td>
		</tr>
		<tr>
			<td>Superfrost Plus microscopy slides</td>
			<td>VWR Scientific</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>22x22 mm coverslips</td>
			<td>VWR Scientific</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 8.4.1</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative whole-mount immunofluorescence analysis of cardiac progenitor populations in mouse embryos

使用不断进步的成像技术, 有助于我们增加对胚胎发育的理解。植入前发育和器官移植是两个领域的研究, 得益于这些进步, 由于数据的高质量, 可以直接从成像前胚胎或活体器官。虽然前胚胎产生的数据具有特别高的空间分辨率, 但后期阶段对三维重建的顺从性较差。为已知的胚胎结构获得高质量的3D 或体积数据, 与命运图或遗传谱系追踪相结合, 可以对发生在胚胎发育过程中的形态发生事件进行更全面的分析。
本议定书描述了一个完整的免疫荧光方法, 允许标记, 可视化, 并定量的祖细胞在发展中的心脏新月, 一个关键的结构在心脏发育过程中形成。这种方法的设计方式是, 可以获得细胞和组织级的信息。利用共焦显微镜和图像处理, 该协议允许三维心脏新月的空间重建, 从而提供了分析特定祖群的定位和组织的能力心脏发育的关键阶段。重要的是, 使用参考抗体可以连续掩蔽心脏新月和随后的定量测量的区域内的新月。本议定书不仅能够对早期心脏发育进行详细的检查, 而且应适用于肠早期节期小鼠胚胎的大多数器官系统。

最终数据和分析的质量很大程度上取决于 (1) 解剖胚胎的完整性和形态学, (2) 使用高特异性抗体, (3) 正确设置成像参数。受损的胚胎会混淆表面生成过程, 阻碍定量分析。正确解剖和分期胚胎的例子如图 2B所示。胚胎可以通过去除卵黄囊进行进一步的解剖, 抗体会经常非。然而, 去除卵黄囊会使胚胎变得不那么健壮, 因此在下游处理过程中应注意。
高质量的抗体和适当的成像设置的结合是实现高信噪比图像的关键。图 2A显示了一个示例图像, 它使用对 Nkx2-5 的抗体作为标记心脏新月的参考标志, 而 GFP 则标记了一个血统跟踪感兴趣的人群 (Foxa2Cre:YFP 心室心血管祖细胞) (见补充原始图像和数据文件的数据)。19此策略允许将给定的总体与整个心脏新月区域进行比较。对于一些实验, 可能有兴趣检查 FHF 和 SHF 的心脏新月的子域。在这种情况下, FHF 和 SHF 可以分别标记为 Hcn4 和 Islet1 (参见参考19 , 用于图像和这些抗体组合的定量分析)。与适当的设置和二级抗体组合, 我们已经成功地成像和分析三血统并发 (即心脏新月, YFP, 和第一或第二 HF)。
无法实现强信号也将限制生成高置信度3D 模型的能力, 因为它将很难从最终的表面去除背景信号。在这些情况下, 应注意调整图像伽玛 (在预处理期间,图 2B, 2C, 2N, 2O) 或表面阈值 (在曲面生成算法中,图 2G, 2H, 2L) 和类似的设置应用于实验中的所有图像。如果不这样做, 将导致不一致的体积数据, 这将是不适当的使用直接比较。通常, 通过大小筛选 (图 2I、 2J) 可以删除背景信号产生的错误曲面, 但从 high-background 图像中这样做会很困难, 而且不会丢失真实的表面碎片。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56446/56446fig1.jpg"> 
图 1: 实验时间线和示意图(A) 整个实验, 从交配到数据分析, 可以在大约两周内进行。胚胎是收集在早上 8th日后交配 (E8.25) 的心脏新月阶段分析 (B)。一旦固定, 胚胎被堵塞和染色的初级和二级抗体之前安装。胚胎使用标准的显微镜幻灯片和片, 用双面胶带作为间隔 (C)。胚胎被放置在一滴防在显示的方向 (c, 上部) 和一个片慢慢地降低到磁带 (c, 更低)。进行共焦成像和图像分析, 以生成高分辨率 z 堆栈图像 (D) 和3D 表面模型 (E)。HF, 头折;CC, 心脏新月;A, 前面;P, 后部;防刻度线为100µm.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56446/56446fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56446/56446fig2.jpg"> 
图 2: 渐进共焦图像处理和3D 曲面生成.(A) 在卷视图中加载的共焦 z 系列。在开始创建曲面算法之前, 会选择参考通道 (Nkx2-5) 和强度和伽玛调整 (C)。选择了感兴趣的区域后 (D), 并选择了曲面细节级别 (E)。初始曲面 (F) 是值的, 用于包括表面上的所有真实信号 (G)。然后执行筛选以移除小背景片段 (H) 以生成最终参照曲面 (I)。通过选择参照曲面 (J), 可以复制和屏蔽比较通道 (YFP) (K)。然后, 在通过相同的步骤序列 (M, N) 生成第二个曲面之前, 对该通道的强度和伽玛进行调整 (L)。每个表面的总体积是自动计算的, 可以定量和可视化地进行比较 (O, 注意, 重叠时表面颜色不合并)。缩放栏 = 100 µm.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56446/56446fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos at JoVE.com

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

在这里, 我们描述了一个协议的全贴装免疫荧光和图像定量容积分析早期小鼠胚胎。我们提出这项技术是一个强有力的方法, 定性和定量评估心脏结构在发展过程中, 并提出它可能会广泛适用于其他器官系统。

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 ...

quantitative-whole-mount-immunofluorescence-analysis-cardiac

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.5K 次观看.  Icahn School of Medicine at Mount Sinai. 在这里, 我们描述了一个协议的全贴装免疫荧光和图像定量容积分析早期小鼠胚胎。我们提出这项技术是一个强有力的方法, 定性和定量评估心脏结构在发展过程中, 并提出它可能会广泛适用于其他器官系统。

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

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

科研

发育生物学

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

多目标显色全安装<em&gt;原位</em&gt;在比较基因表达域杂交<em&gt;果蝇</em&gt;胚胎

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

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 ...

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 ...

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, ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

小鼠心脏场特定心脏祖细胞的体外生成

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...