Name: analysis of congenital heart defects in mouse embryos using qualitative and quantitative histological methods
Uploaded: 2020-03-10T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods at JoVE.com

阿吉雷实验室由美国国家卫生研究院国家心肺血液研究所支持，奖励编号为K01HL135464，美国心脏协会奖励编号为19IPLOI34660342。

本文引用的实验中使用的所有动物都使用密歇根州立大学机构动物护理和使用委员会 （IACUC） 的动物护理指南进行处理。
1. C57BL6/J小鼠用于胚胎生产的时配交配
<ol><li>一旦小鼠达到繁殖年龄（6-8周），将它们以兔子繁殖的形式（即，每只雄性两只雌性）放在一起。把它们安排在下午或晚上的某个时候繁殖。 注：老鼠通常在1小时内繁殖后，灯已经关闭在他们的住房设施内?...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+toward+heart+regeneration:+Stem+cells+and+beyond.">Reprogramming toward heart regeneration: Stem cells and beyond.</a> Cell Stem Cell. 12 (3), 275-284 (2013).</li><li>Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+or+breaking+the+heart:+from+lineage+determination+to+morphogenesis.">Making or breaking the heart: from lineage determination to morphogenesis.</a> Cell. 126 (6), 1037-1048 (2006).</li><li>Heyne, G. 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该协议探讨了胚胎心脏发育分析所涉及的技术。这种方法的一些局限性是准备技术所需的物理灵巧性，这可能需要练习，以及显微镜成像技术。如果在低温塔获得的切片是凌乱的，血氧林和欧辛染色将不清楚，或者如果在显微镜上拍摄的图像照明不良，则与 ImageJ 一起使用的方法将不起作用。ImageJ 软件的阈值功能的一个限制是它选择位于阈值一端的像素，具体取决于像素值。因此，在计算中错误?...

CHD是人类最常见的先天缺陷类型，是出生缺陷相关死亡的主要原因1、2、3、4、5、6。虽然约90%的新生儿在CHD存活下来，但多年来经常与重大的发病率和医疗干预有关，给病人的生活和医疗系统带来沉重的负担。除了纯遗传因素外，CHD的病因还知之甚少。根据美国心脏协会和其他来源2、3、4、11，不明原因占所有CHD病例的56-66%。众所周知的因素包括基因突变、CNV、诺诺单核苷酸变异和阿非普肽。人们怀疑环境和饮食因素也是导致CHD的重要来源，流行病学研究表明，将母亲的生活方式2、12、经济剥夺和种族13联系起来，并通过研究饮食因素，如叶酸11，14和生物活性脂视网膜酸15，16。研究CHD和其他心血管缺陷的机制和原因，对于制定预防策略和新颖的治疗选择（1、4、17、18、19）非常重要。
发育中的小鼠是研究哺乳动物CHD的基石模型。然而，一些方法和分析，如解剖保存心脏形态，分析发育阶段，以及识别CHD相关缺陷，对于对分析鼠心的新科学家来说，可能令人望而生畏。该协议中描述的方法的目标是为这些流程提供定性和定量指南。因此，在此协议中，我们解释如何进行定时交配以产生所需妊娠阶段的胚胎，解剖怀孕的雌性，以恢复完整的心脏（包括相关组织，如流出道），心脏固定和准备冷冻切片，基本组学方法，常见心脏缺陷的定量分析，以及心肌压实的定性分析，是某些类型的CHD的常见前体表型。

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			<td>15 mL Conical Tube(s)</td>
			<td>Fisher Scientific</td>
			<td># 1495970C</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J Mice</td>
			<td>Jackson Labs</td>
			<td>C57BL/6J - stock 000664</td>
			<td></td>
		</tr>
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			<td>Coplin Staining Jars (x6)</td>
			<td>VWR Scientific</td>
			<td># 25457-006</td>
			<td></td>
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			<td>Coverslips 24X50MM #1.5</td>
			<td>VWR Scientific</td>
			<td># 48393-241</td>
			<td></td>
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			<td>Cryostat - Leica CM3050S</td>
			<td>Leica</td>
			<td>N/A</td>
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			<td>Dissecting Dish(s)</td>
			<td>Fisher Scientific</td>
			<td># 50930381</td>
			<td></td>
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			<td>Dumont #5 - Fine Forceps (x2)</td>
			<td>Fine Science Tools</td>
			<td># 11254-20</td>
			<td></td>
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			<td>Eosin Y Solution</td>
			<td>Millipore Sigma</td>
			<td># HT110116-500ML</td>
			<td></td>
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			<td>Ethyl Alcohol (Pure, 200 proof)</td>
			<td>Fisher Scientific</td>
			<td># BP2818-500</td>
			<td></td>
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			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Millipore Sigma</td>
			<td># E9884-100G</td>
			<td></td>
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			<td>Eukitt</td>
			<td>Millipore Sigma</td>
			<td># 03989-100ML</td>
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			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td># 14060-10</td>
			<td></td>
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			<td>Fluorescent Stereo Microscope Leica M165 FC</td>
			<td>Leica</td>
			<td>N/A</td>
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			<td>Glycine</td>
			<td>Millipore Sigma</td>
			<td># 410225-250G</td>
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			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td># 11052-10</td>
			<td></td>
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			<td>Graphpad Prism 8 Software</td>
			<td>Graphpad</td>
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			<td>ImageJ Software</td>
			<td>ImageJ</td>
			<td></td>
			<td></td>
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			<td>Kimwipes</td>
			<td>Fisher Scientific</td>
			<td># 06666A</td>
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			<td>Mayer&#39;s hematoxylin solution</td>
			<td>Millipore Sigma</td>
			<td># MHS16-500ML</td>
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			<td>Micropipette tip(s) - p200</td>
			<td>Fisher Scientific</td>
			<td># 02707448</td>
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			<td>Microsoft Excel Software</td>
			<td>Microsoft</td>
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			<td>OCT Compound</td>
			<td>VWR Scientific</td>
			<td># 102094-106</td>
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			<td>Olympus CkX53 Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
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			<td>Paint Brushes (at least 2)</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Paraformaldehyde</td>
			<td>VWR Scientific</td>
			<td># 0215014601</td>
			<td>Make into 4% solution (dissolved in PBS)</td>
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			<td>Pasteur pipette(s)</td>
			<td>Fisher Scientific</td>
			<td># 13-711-7M</td>
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			<td>Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td># 15140122</td>
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			<td>Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td># 70011044</td>
			<td>Dilute from 10x to 1x before using</td>
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			<td>Scale</td>
			<td>Mettler Toledo</td>
			<td># MS1602TS</td>
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			<td>Scale</td>
			<td>Mettler Toledo</td>
			<td># MS105</td>
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			<td>Scalpel Handle #3</td>
			<td>VWR Scientific</td>
			<td># 10161-918</td>
			<td></td>
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			<td>Scalpel Blades</td>
			<td>VWR Scientific</td>
			<td># 21909-612</td>
			<td></td>
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			<td>Square Mold</td>
			<td>VWR Scientific</td>
			<td># 100500-224</td>
			<td>For OCT molds</td>
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			<td>Sucrose</td>
			<td>Millipore Sigma</td>
			<td># S9378-500G</td>
			<td></td>
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			<td>Superfrost Plus Slides</td>
			<td>Fisher Scientific</td>
			<td># 1255015</td>
			<td></td>
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			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
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			<td>Tissue-Tek Accu-Edge Disposable Microtome Blades</td>
			<td>VWR Scientific</td>
			<td># 25608-964</td>
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			<td>Travel Scale</td>
			<td>Acculab</td>
			<td>VIC 5101</td>
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			<td>Xylene</td>
			<td>Millipore Sigma</td>
			<td>214736-1L</td>
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analysis of congenital heart defects in mouse embryos using qualitative and quantitative histological methods

先天性心脏缺陷（CHD）是人类最常见的先天缺陷类型，影响多达1%的活产。然而，CHD的根本原因仍然缺乏理解。发育中的小鼠是研究CHD的宝贵模型，因为小鼠和人类之间的心脏发育程序高度保守。该协议详细介绍了如何产生所需妊娠阶段的小鼠胚胎、分离和保存心脏的方法，以及通过组织学识别常见CHD类型的定量方法（例如心室隔膜）缺陷、心房隔膜缺陷、专利导管动脉、定量通体测量方法，以测量常见的肌肉压实现象类型。这些方法阐明了样品制备、收集和分析所涉及的所有步骤，使科学家能够正确和repad测量CHD。

肌肉压实指数在两种不同环境下发育的心脏（对照组和实验组）之间进行了比较。这些协议用于定量分析肌肉组织的压实，从而允许进行统计分析。与在非实验条件下发育的胚胎相比，实验心脏的肌肉压实作用显著减少。
如果观察时间似乎不准确，胚胎就被丢弃了。虽然胚胎发育迟缓可能是实验治疗的结果，而且发育进展也?...

Watch this Scientific Journal Video about Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods at JoVE.com

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

在此协议中，我们描述了定性和定量分析与先天性心脏缺陷相关的小鼠发育表型的程序。

使用定性和定量组织方法对小鼠胚胎先天性心脏缺陷的分析

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

该协议标准化了新的组织分析技术，以便可以更广泛地被研究先天性心脏缺陷的其他组使用。由于定性和定量分析技术各有优缺点，这种方法同时使用两者来解决最广泛的实验问题。这些技术对心脏病诊断方法有积极影响。例如，病理学家可以应用我们的显微心脏组织分析技术，从活检样本中诊断人类心脏病。该协议可用于发展心脏病学或一般心脏病学研究。然而，这些方法也提供了各种有用的技术，可以应用于任何组织分析。为了收集胚胎第15至15.5天的心脏，固定怀孕的大坝的四肢，并围绕尿道开始到胸骨，小心地沿着躯干做一个我形切口。在铲取运动中使用钳子，轻轻地将子宫从腹腔中抬出，必要时用细钳抓住子宫的肤浅组织，以帮助提取。用剪刀从腹部释放子宫。将子宫浸泡在冰冷的PBS中后，将器官固定到解剖盘中。用剪刀将子宫切成单独的部分，每个部分包含一个单独的胚胎。使用两对细钳子，轻轻地将胚胎从子宫组织中剥离出来，并将它们放在含有四摄氏度PBS-EDTA的新培养皿中。为了收集心脏，将盘子放在解剖显微镜下，并使用两对细钳将胚胎置于子宫内位置。稳定进入胸部后，使用第二对细钳的尖点，沿着胚胎的胸骨切口，在锁骨到肚脐之间延伸。使非常精细和肤浅的切口，同时逐渐工作到胸腔内侧，打开胚胎的腹部。当心脏变得可见时，用一对钳子将躯干稍微挤到肋骨笼上，将肋骨剥开。使用一对钳子的两侧，轻轻抓住肺血管，而不分离组织，将心脏从腔中拉出。在新鲜PBS-EDTA中清洁心脏一至两分钟，然后将组织固定在4%的甲醛中45至60分钟。然后，在PBS中每次洗5至10分钟，并辅以甘氨酸，洗心脏三次。在将心脏储存在PBS中摄氏4度之前。为了准备心脏的冷冻，将样品放入30%蔗糖和PBS的管中，在4摄氏度下24至40小时。当心脏沉入管底时，使用具有修改尖端的巴斯德移液器小心地将每颗心转移到一块无绒擦拭上。短暂空气干燥后，将每个样品放入单独的最佳切割温度模具中，以所需的方向进行切割。将组织淹没在最佳切割温度介质中，无气泡。然后，将霉菌在20摄氏度下储存几天，然后在80摄氏度下冷冻组织至少24小时，然后进行冷冻。要获取样品的低温分段，请使用新鲜的最佳温度切割介质将感兴趣的组织块连接到低温恒温器的夹头上，并允许该介质冻结，直到其不透明为白色。插入新刀片并调整组织块与刀片之间的距离，以便获得截面。在 10 微米切片中修剪块，直到到达感兴趣点。当可以观察到组织时，使用小型平面画笔以连续切割运动轻轻地将切片拉到支架上。一旦组织被完全切开，使用细节刷拍下来的切片平对台。在取下详细笔刷并完成切片之前，将截面保持到位约 20 到 30 秒。使用两个画笔轻轻地将切片压平，防止任何滚动，并按住组织部分对舞台。20 到 30 秒后，翻转该部分并再次将其压平到舞台上，然后放置一个静电滑动，使切片足够接近，以吸引切片并粘附到幻灯片上，而无需将幻灯片触摸到舞台上。要评估心脏组织部分是否存在常见心脏缺陷，使用配备相机的显微镜，以 5 到 10 倍和 40 倍的放大倍率获得图像。对于心肌压实分析，在适当的图像分析软件程序中打开感兴趣的组织，将图像设置为 8 位。要设置图像的阈值，请单击"图像、调整"和"阈值"，然后移动顶部栏以调整阈值，直到仅选择背景像素。接下来，使用多边形选择工具在组织周围绘制感兴趣的区域，然后单击"分析、设置测量、面积和限制阈值"。单击"分析"和"测量"可测量高亮显示像素的面积，以获取负空间区域的值。要测量整个感兴趣区域的区域，请单击"分析"、设置度量和取消选择阈值限制。然后，选择"分析和测量"以测量所选感兴趣区域的整个区域。要计算肌肉组织的面积，请从感兴趣区域的负空间区域中减去"负空间"。然后将肌肉组织区域除以"感兴趣区域"来计算肌肉压实和X.从收获的胚胎心脏中染色组织切片，提供足够的对比度来分出组织边缘，但并没有使细胞特征难以区分。在这个具有代表性的分析中，观察到肌肉压实在实验心脏中相对于在非实验条件下发育的胚胎显著降低。这些样品很有价值，需要很长时间才能创建。在尸检和低温统计剖切期间，请务必有意执行每一个动作。保存准甲醛中的组织可保持抗原活性，允许样品用于亚米诺氟素染色等测试，帮助确定形态评估期间发现的病理原因。在我们的实验室中，我们获得了实验治疗的定量表示，可以帮助我们确定不同治疗如何影响我们的样品。甲状醛可以修复它接触的任何组织。注意始终戴上手套，尽量减少暴露的皮肤，并在使用这种化学品时在薄膜罩中工作。

Research

JoVE Journal

Developmental Biology

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

使用小鸡GFP 神经管移植和碳水化合物染料 DiI 注射，在鸡胚胎内对神经囊细胞和血管进行双重标记

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

科研

发育生物学

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

对比成像的小鼠胚胎使用高频超声

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic 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 ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

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

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 ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

在清除胚胎心脏冠状血管分析

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

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

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 ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

用高频超声 (30/45 mhz) 系统对胎儿小鼠进行心血管成像

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...