Name: quantitative whole-mount immunofluorescence analysis of cardiac progenitor populations in mouse embryos
Uploaded: 2017-10-12T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos at JoVE.com

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 <...

<ol>
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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?...

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。
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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。

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

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

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

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

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

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 ...

Whole Mount Immunohistochemistry in 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. ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

使用全载免疫作用化学和三维重建对咽弓动脉的可视化与分析

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...