Name: whole-mount immunofluorescence staining, confocal imaging and 3d reconstruction of the sinoatrial and atrioventricular node in the mouse
Uploaded: 2020-12-22T15:00:00.000+00:00
Duration: 5 min 16 s
Description: Watch this Scientific Journal Video about Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse at JoVE.com

这项工作得到了中国国家留学基金委（CSC，对R. Xia），德国心血管研究中心（DZHK;81X2600255给S. Clauss，81Z0600206给S. Kääb），科罗纳基金会（S199/10079/2019给S. Clauss），SFB 914（项目Z01给H. Ishikawa-Ankerhold和S. Massberg，项目A10到C. Schulz），ERA-NET心血管疾病（ERA-CVD;01KL1910给S. Clauss）和Heinrich-and-Lotte-Mühlfenzl Stiftung（给S. Clauss）的支持。资助者在手稿准备中没有任何作用。

动物护理和所有实验程序均按照慕尼黑大学动物护理和伦理委员会的指导方针进行，对小鼠进行的所有程序均获得德国慕尼黑巴伐利亚州政府的批准（ROB-55.2-2532.Vet_02-16-106，ROB-55.2-2532.Vet_02-19-86）。C57BL6 / J小鼠从杰克逊实验室购买。
注意: 图1 显示了实验所需的仪器。图2显示了大体心脏解剖结构的 图 示。 图 3 显示了 SAN 和 AVN 在成年小鼠心脏中的位置。 图4 显示了在共聚焦显微镜上加载的制备样品。
1. 准备工作
<ol><li>通过在 30 mL PBS 中稀释 10 mL 的 16% 甲醛溶液来制备 4% 甲醛溶液 （4% PFA）。</li>
	<li>通过将 15 g 和 30 g 蔗糖粉分别溶解到 100 mL PBS 中来制备 15% 蔗糖溶液和 30% 蔗糖溶液。蔗糖粉完全溶解后，在4°C储存之前，使用0.2μm注射器过滤器过滤溶液。</li>
	<li>准备3%-4%琼脂糖凝胶，将3 g或4 g琼脂糖粉和100 mL 1x TAE（从50x TAE储备溶液稀释）加入烧杯中。将烧杯放在磁力搅拌器上煮沸，直到琼脂糖完全溶解。</li>
	<li>通过将30mL琼脂糖凝胶（3%-4%）轻轻倒入直径为100毫米的培养皿中来准备解剖盘。将培养皿放在室温下的工作台上冷却并变硬。</li>
	<li>根据 表1中提供的配方制备封闭溶液和洗涤溶液。准备使用当天准备的所有溶液。不建议长期储存。</li>
	<li>通过在冷（4°C）1x PBS中稀释抗体溶液来制备抗体溶液，保护稀释液免受光照（例如，通过包裹铝箔），并将稀释液保持在冰上直至使用。孵育前不久稀释抗体。避免将稀释的抗体留在室温下。 注意:应通过进行常规免疫荧光染色，用可比较的组织样品测试适当的抗体稀释度。在这里，我们通过染色厚度为10μm的冷冻切片来测试抗体。</li>
</ol>2. 器官摘取和组织准备
<ol><li>通过将小鼠放入连接到异氟醚蒸发器的孵育室中来麻醉小鼠。将蒸发器设置为提供4-5%的异氟醚（分别为96-95%的氧气）。 注意:通过轻轻滚动腔室直到鼠标仰卧，通过失去姿势反应和矫正反射来确认完全麻醉。</li>
	<li>将鼠标仰卧在手术台上。将鼠标鼻子放入连接到改良贝恩回路的麻醉面罩中，其内管连接到异氟烷蒸发器。为了维持麻醉，在氧气中使用1-2%异氟醚，流速为1L / min。通过面罩的外管清除小鼠身上多余的麻醉蒸气，并通过一罐活性炭吸入，活性炭吸收多余的麻醉气体。</li>
	<li>达到完全麻醉后，注射芬太尼镇痛（0.1 μg / 25 g体重ip.p.）。</li>
	<li>当无法检测到脚趾捏反射时，使用虹膜剪刀从颈静脉到联合进行清晰的切口，以去除皮毛和皮肤。用虹膜剪刀从左到右在肋骨下方再剪一次，小心地打开腹部。</li>
	<li>使用弯曲的镊子稍微切割剑突，以便在不伤害任何器官的情况下从左到右切割横膈膜。用虹膜剪刀在两侧的内侧腋窝线上切割肋骨，使其颅骨翻转并允许进入心脏。</li>
	<li>使用虹膜剪刀在横膈膜水平处切割下腔静脉和下降胸主动脉。用27 G针刺穿心尖区域的心脏，然后小心地将针头推入左心室（LV）。将 5-10 mL 冰冷的 PBS 轻轻注入左心室以灌注心脏。 注意:心脏的颜色应从红色变为灰色，表明PBS灌注成功。</li>
	<li>用镊子小心地抬起心脏的顶点，以切割大血管并取出心脏。</li>
	<li>通过尽可能远离心脏切开大动脉和静脉来切除心脏，以避免对上腔静脉 （SVC） 造成任何损害。保存 SVC，因为它将在以后的处理中充当重要的地标。</li>
	<li>心脏取出后，关闭异氟醚蒸发器。</li>
	<li>在解剖显微镜下将心脏放入装满冰冷PBS的解剖盘中。确定心脏的左/右和前/后，将心脏的前部放在培养皿底部（以暴露位于后方的大血管）转动心脏。</li>
	<li>通过将小针穿过顶点和左心耳（LAA）放入解剖盘底部的琼脂糖中来固定心脏（图2A）。使用细镊子和剪刀，小心地去除上腔静脉和下腔静脉（IVC）（例如肺，脂肪，心包）周围的非心脏组织，以暴露腔间区域（图3A）。</li>
	<li>通过用微型剪刀平行切割心室和心房之间的凹槽来去除大部分心室。保留一小部分心室组织，以便以后在Plexiglas环上加载。</li>
	<li>对于固定和脱水，将样品（包含心房，SVC和IVC）放入4%PFA中，在4°C下过夜。</li>
	<li>第二天，将心脏转移到4°C下的15%蔗糖溶液中24小时。</li>
	<li>第二天，将心脏转移到30%蔗糖溶液中，在4°C下24小时。 注意:对于步骤2.13，2.14和2.15中的固定和脱水，样品保持在4°C，无需进一步搅拌或摇摆。</li>
</ol>3. 全安装免疫荧光染色
<ol><li>用PBS稀释的1%Triton X-100洗涤心脏，并在封闭溶液（表1）中封闭并透化过夜，温度为4°C。</li>
	<li>将心脏置于1.5mL管中，并与兔抗小鼠连接蛋白-43（稀释1:200）和大鼠抗小鼠HCN4（稀释1:200）抗体在4°C下用封闭溶液稀释7天孵育。 注意:在使用数据表作为方向之前，应测试一抗的最佳浓度。其他感兴趣的抗原也可以在此步骤中染色，只要主要抗原的宿主物种与其他抗原不同。较高浓度的Triton X-100可能有助于获得更有效的抗体染色，如10之前所示，但浓度可以单独确定。</li>
	<li>7天后，使用移液器除去含有一抗的溶液，并用1%Triton X-100溶液洗涤心脏3次（每次在室温下在轨道振荡器上洗涤1小时）。</li>
	<li>洗涤后，将心脏=与Alexa Fluor 488山羊抗大鼠IgG（稀释度为1:200）和Alexa Fluor 647山羊抗兔IgG（稀释度为1:200）在4°C下孵育7天。</li>
	<li>7天后，使用移液器除去所有含有二抗的溶液。然后使用洗涤溶液（表1）洗涤心脏3次（每次在室温下在轨道振荡器上洗涤1小时）。</li>
	<li>要对细胞核进行染色，请将心脏在 DAPI 溶液 （10 μg/mL） 中在 4 °C 下孵育过夜。</li>
	<li>第二天，用洗涤溶液洗涤心脏3次（每次在室温下在轨道摇床上洗涤1小时）。 注意:对于步骤3.1，3.2，3.4和3.6中的封闭，透化，抗体孵育和DAPI染色，将样品保持在4°C的稳态，无需搅拌。染色的组织可以在4°C下用洗涤溶液完全覆盖并避光几天，直到在共聚焦显微镜上成像。</li>
</ol>4. 共聚焦显微镜
<ol><li>准备Plexiglas环并填充橡皮泥（图1）。在橡皮泥的中心形成一个小凹槽，用于装载心脏。在步骤4.4中，做一个浅凹槽，因为这更容易获得平坦的成像平面，并调整空间并避免样品和盖玻片之间的气泡。</li>
	<li>使用相同的心脏样本按顺序对 SAN 和 AVN 进行成像。对于 SAN 成像，直接加载心脏，而对于 AVN 成像，在之前进行显微切割。<ol><li>SAN 映像<ol><li>通过解剖标志物识别SAN:它位于腔间区域（上腔静脉和下腔静脉之间的区域）内的心脏背侧。终突（CT）是SAN和RAA之间的肌肉条纹。</li>
			<li>将心脏放入橡皮泥槽中，心脏背面朝上。将PBS添加到心脏上以置换腔内的所有空气，直到心脏完全被PBS覆盖（通常几滴PBS就足够了）。</li>
			<li>在解剖显微镜下，将RAA，LAA和左心室的其余部分轻轻按入橡皮泥中以固定心脏。确保可以清楚地看到整个腔间区域和末端吼肘（未被橡皮泥覆盖）。将成像的区域如图 3A所示。</li>
		</ol></li>
		<li>AVN 成像<ol><li>完成SAN成像后，重新收集相同的样品，随后用于AVN成像。对于AVN显微切割，将心脏=放在解剖显微镜下的解剖盘上。</li>
			<li>使心脏右侧朝上（包括右心室的其余部分）定向。将针穿过LV自由壁的其余部分（现在位于底部）以固定组织（图2B）。</li>
			<li>通过三尖瓣和上腔静脉向上切开剩余的无右心室壁。然后将 RV 和 RA 翻转开，露出室间和房间隔。（图3B） 注意:注意不要损伤冠状窦（CS），因为它是找到Koch三角形的重要解剖标志，然后可以定位AVN。CS在心脏后侧的左房室沟中横向运行，CS口开口位于IVC和房间隔下部的三尖瓣之间（图3A 和B）。</li>
			<li>识别可在右心房心内膜表面找到的 Koch 三角形，前边界为三尖瓣 （TV） 隔叶的铰链线，后方与 Todaro 肌腱接壤。基底由冠状窦的孔形成（图3B）。由于这是AVN成像的目标区域，因此需要清晰可见。</li>
			<li>将心脏转移到Plexiglas环内的橡皮泥槽中，科赫三角形明显暴露（即没有被橡皮泥覆盖）。轻轻地将科赫三角形周围的组织按入橡皮泥中以固定样品。将PBS添加到心脏上以置换腔内的所有空气，直到心脏完全被PBS覆盖（通常几滴PBS就足够了）。</li>
		</ol></li>
	</ol></li>
	<li>将硅胶涂在Plexiglas环的边缘，以便用盖玻片覆盖填充橡皮泥的Plexiglas环内的心脏。</li>
	<li>轻轻按压橡皮泥的背面以挤出PBS的一部分并将心脏连接到盖玻片上，同时避免成像区域内出现任何气泡。 注意: 确保在按压橡皮泥的背面时未折叠和覆盖感兴趣的区域。没有样品过度压缩的平面成像平面对于保存解剖结构和样品的正确共聚焦成像是必要的。对于 SAN，重要的是要确保腔间区域清晰暴露。对于AVN，科赫三角形应该完全暴露。</li>
	<li>将整个安装染色样品倒置在共聚焦显微镜的平台上。附在盖玻片上的暴露的SAN/AVN现在位于显微镜平台的底部（图4）。 注意:要拍摄图像，我们使用带有Airyscan单元的卡尔蔡司LSM800和软件ZEN 2.3 SP1黑色。</li>
	<li>选择板"BP420-480 + LP605"来激发Alexa Fluor 647共轭抗HCN4。改变主增益范围为650-750。</li>
	<li>使用 切片扫描 功能获取整个SAN和AVN区域的概述图像。然后，通过单击并在将要扫描的感兴趣区域周围的概述图像上添加一个正方形来选择 HCN4 阳性区域。</li>
	<li>检查共聚焦显微镜其余通道（Alexa Fluor 488和DAPI）的参数，包括板和主增益，并按照步骤4.6中所述进行设置。</li>
	<li>从样本的顶部到底部缓慢调整焦点以预览整个样品，并为 Z 堆栈范围设置" 第一个 "和 "最后一个 "。根据光学样品的厚度为 Z 堆栈设置最佳间隔。我们使用20倍物镜，0.8-1μm作为Z堆栈的间隔。</li>
	<li>正确设置所有参数后，扫描 SAN 和 AVN 的整个区域。</li>
	<li>使用软件（例如，Imaris 版本 8.4.2）对图像进行 3D 重建。<ol><li>选择 图像处理 |基线减法 以去除背景染色。</li>
		<li>在所选通道和感兴趣区域的设置上选择曲面创建以进行处理。</li>
	</ol></li>
</ol>

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V., Morales, M. J., Campbell, D. L., Steenbergen, C., Strauss, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+distribution+of+voltage-gated+ion+channels+in+ferret+sinoatrial+node.">Expression and distribution of voltage-gated ion channels in ferret sinoatrial node.</a> Physiological Genomics. 42 (2), 131-140 (2010).</li><li>Mesirca, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+arrhythmia+induced+by+genetic+silencing+of+'funny'+(f)+channels+is+rescued+by+GIRK4+inactivation.">Cardiac arrhythmia induced by genetic silencing of 'funny' (f) channels is rescued by GIRK4 inactivation.</a> Nature Communication. 5, 4664 (2014).</li><li>Liang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCN4+dynamically+marks+the+first+heart+field+and+conduction+system+precursors.">HCN4 dynamically marks the first heart field and conduction system precursors.</a> Circulation Research. 113 (4), 399-407 (2013).</li><li>Verheule, S., Kaese, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+diversity+in+the+heart:+insights+from+transgenic+mouse+models.">Connexin diversity in the heart: insights from transgenic mouse models.</a> Frontiers in Pharmacology. 4, 81 (2013).</li><li>van der Velden, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+gap+junctions+and+connexins:+their+role+in+atrial+fibrillation+and+potential+as+therapeutic+targets.">Cardiac gap junctions and connexins: their role in atrial fibrillation and potential as therapeutic targets.</a> Cardiovascular Research. 54 (2), 270-279 (2002).</li></ol>

提交人声明他们没有利益冲突。

传统上，心脏解剖学研究使用薄的组织学切片11。然而，这些方法不保留传导系统的三维结构，因此仅提供2D信息。此处描述的全卡口免疫荧光染色方案可以克服这些限制，并且可以常规用于SAN和AVN成像。
与需要石蜡包埋、切片和抗原修复的常规免疫组织化学等标准方法相比，全卡口方法有利于解决SAN和AVN的精确3D定位和形态，并检查与周围组织的关系，因?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62058">Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse</a>. The Authors section was updated from:
Ruibing Xia1,2,3 
Julia Vlcek1,2 
Julia Bauer1,2,3 
Stefan K&#228;&#228;b1,3 
Hellen Ishikawa-Ankerhold1,2 
Dominic Adam van den Heuvel1,2 
Christian Schulz1,2,3 
Steffen Massberg1,2,3 
Sebastian Clauss1,2,3 
1University Hospital Munich, Department of Medicine I, Ludwig Maximilian University Munich 
2Walter Brendel Center of Experimental Medicine, Ludwig Maximilian University Munich 
3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance
to:
Ruibing Xia1,2,3 
Julia Vlcek1,2 
Julia Bauer1,2,3 
Stefan K&#228;&#228;b1,3 
Hellen Ishikawa-Ankerhold1,2 
Dominic Adam van den Heuvel1,2 
Christian Schulz1,2,3 
Steffen Massberg1,2,3 
Sebastian Clauss1,2,3 
1University Hospital Munich, Department of Medicine I, Ludwig Maximilians University (LMU) Munich 
2Institute of Surgical Research at the Walter Brendel Center of Experimental Medicine, University Hospital Munich, Ludwig Maximilians University (LMU) Munich 
3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62058">Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse</a>. The Authors section was updated.

Erratum: Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

心律失常是影响数百万人的常见疾病，是全世界发病率和死亡率高的原因。尽管在治疗和预防方面取得了巨大进展，例如心脏起搏器的发展，但心律失常的治疗仍然具有挑战性，主要是由于对潜在疾病机制的了解非常有限1，2，3。更好地了解心律失常的正常电生理学和病理生理学可能有助于在未来开发新颖、创新和因果治疗策略。此外，为了全面研究心律失常发生，重要的是定位和可视化小鼠等动物模型中的特定心脏传导系统，因为小鼠广泛用于电生理学研究。
心脏传导系统的主要部分是窦房结（SAN），电脉冲在专门的起搏器细胞中产生，以及房室结（AVN），这是心房和心室之间唯一的电连接4。每当SAN和AVN的电生理特性改变时，就会发生病态窦房结综合征或房室传导阻滞等心律失常，这可能导致血流动力学恶化，晕厥甚至死亡，从而强调了SAN和AVN在电生理学和心律失常发生中的重要作用5。
对SAN或AVN的全面研究需要对这两种结构进行精确的定位和可视化，最好是在它们的生理环境中。然而，由于它们的体积小且在工作心肌中的位置很小，如果没有建立清晰的宏观可见结构，研究SAN和AVN的解剖学和电生理学具有挑战性。解剖标志可用于粗略识别包含 SAN 和 AVN6，7，8 的区域。简而言之，SAN位于右心房的腔间区域，毗邻肌肉末端嵴（CT），AVN位于由三尖瓣，冠状窦的开口和Todaro的肌腱建立的Koch三角形内。到目前为止，这些解剖标志主要用于定位，去除然后研究SAN和AVN作为单个结构（例如，通过常规组织学）。然而，为了更好地了解SAN和AVN的复杂电生理学（例如，工作心肌相邻细胞的调节作用），有必要研究生理3D环境中的传导系统。
全卡口免疫荧光染色是一种用于原 位 研究解剖结构同时保持周围组织完整性的方法9。利用共聚焦显微镜和图像分析软件，可以使用荧光标记的抗体可视化SAN和AVN，这些抗体靶向在这些区域中特异性表达的离子通道。
以下协议解释了为SAN和AVN显微镜定位和可视化执行成熟的全安装染色方法的必要步骤。具体来说，该协议描述了如何（1）通过解剖标志定位SAN和AVN，以制备这些样品以进行染色和显微镜分析（2）对参考标记物HCN4和Cx43进行全卡口免疫荧光染色（3）制备SAN和AVN样品用于共聚焦显微镜（4）以执行SAN和AVN的共聚焦成像。我们还描述了如何修改该协议以包括对周围工作心肌或非肌细胞（如自主神经纤维）进行额外染色，从而可以彻底研究心脏内的心脏传导系统。

<table><tbody><tr><td>&lt;strong&gt;Anesthesia&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Isoflurane vaporizer system&amp;nbsp;</td><td>Hugo Sachs Elektronik</td><td>34-0458, 34-1030, 73-4911, 34-0415, 73-4910</td><td>Includes an induction chamber, a gas evacuation unit and charcoal filters</td></tr><tr><td>Modified Bain circuit</td><td>Hugo Sachs Elektronik</td><td>73-4860</td><td>Includes an anesthesia mask for mice</td></tr><tr><td>Surgical Platform</td><td>Kent Scientific</td><td>SURGI-M</td><td></td></tr><tr><td>&lt;strong&gt;&lt;em&gt;In vivo&lt;/em&gt; instrumentation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fine forceps</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Iris scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Spring scissors</td><td>Fine Science Tools</td><td>91500-09</td><td></td></tr><tr><td>Tissue forceps</td><td>Fine Science Tools</td><td>11051-10</td><td></td></tr><tr><td>Tissue pins</td><td>Fine Science Tools</td><td>26007-01</td><td>Could use 27G needles as a substitute</td></tr><tr><td>&lt;strong&gt;General lab instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Orbital shaker</td><td>Sunlab</td><td>D-8040</td><td></td></tr><tr><td>Magnetic stirrer</td><td>IKA&amp;nbsp;</td><td>RH basic</td><td></td></tr><tr><td>Pipette,volume 10 &amp;micro;L, 100 &amp;micro;L, 1000 &amp;micro;L</td><td>Eppendorf</td><td>Z683884-1EA</td><td></td></tr><tr><td>Microscopes</td><td></td><td></td><td></td></tr><tr><td>Dissection stereo- zoom microscope&amp;nbsp;</td><td>VWR</td><td>10836-004</td><td></td></tr><tr><td>Laser Scanning Confocal microscope</td><td>Zeiss</td><td>LSM 800</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Imaris 8.4.2</td><td>Oxford instruments</td><td></td><td></td></tr><tr><td>ZEN 2.3 SP1 black</td><td>Zeiss</td><td></td><td></td></tr><tr><td>&lt;strong&gt;General Lab Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.2 &amp;micro;m syringe filter</td><td>Sartorius</td><td>17597</td><td></td></tr><tr><td>100 mm petri dish</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>27G needle</td><td>BD Microlance 3</td><td>300635</td><td></td></tr><tr><td>50 ml Polypropylene conical Tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>5ml Syringe</td><td>Braun</td><td>4606108V</td><td></td></tr><tr><td>Cover slips</td><td>Thermo Scientific</td><td>7632160</td><td></td></tr><tr><td>Eppendorf Tubes</td><td>Eppendorf</td><td>30121872</td><td></td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.5 M EDTA</td><td>Sigma</td><td>20-158</td><td>Components of TEA</td></tr><tr><td>16% Formaldehyde Solution</td><td>Thermo Scientific&amp;nbsp;</td><td>28908</td><td>use as a 4% solution&amp;nbsp;</td></tr><tr><td>Acetic acid</td><td>Merck</td><td>100063</td><td>Components of TEA</td></tr><tr><td>Agarose</td><td>Biozym</td><td>850070</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A2153-100G</td><td></td></tr><tr><td>DPBS (1X) Dulbecco&#039;s Phosphate Buffered Saline</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Normal goat serum</td><td>Sigma</td><td>NS02L</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S1888-1kg</td><td></td></tr><tr><td>Tris-base</td><td>Roche</td><td>TRIS-RO</td><td>Components of TEA</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-250ml</td><td>Diluted to 1% in PBS</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P2287-500ml</td><td></td></tr><tr><td>&lt;strong&gt;Drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fentanyl 0.5&amp;nbsp;mg/10&amp;nbsp;mL</td><td>Braun Melsungen</td><td></td><td></td></tr><tr><td>Isoflurane 1 mL/mL</td><td>Cp-pharma</td><td>31303</td><td></td></tr><tr><td>Oxygen 5 L</td><td>Linde</td><td>2020175</td><td>Includes a pressure regulator</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Goat anti-Rabbit IgG Alexa Fluor&amp;nbsp;488&amp;nbsp;</td><td>Cell Signaling Technology</td><td>#4412</td><td>diluted to 1:200</td></tr><tr><td>Goat anti-Rat IgG Alexa Fluor 647</td><td>Invitrogen</td><td>#A-21247</td><td>diluted to 1:200</td></tr><tr><td>Hoechst 33342, Trihydrochloride, Trihydrate (DAPI)</td><td>Invitrogen</td><td>H3570</td><td>diluted to 1:1000</td></tr><tr><td>Rabbit Anti-Connexin-43</td><td>Sigma</td><td>C6219</td><td>diluted to 1:200</td></tr><tr><td>Rat anti-HCN4 (SHG 1E5)</td><td>Invitrogen</td><td>MA3-903</td><td>diluted to 1:200</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plexiglass ring</td><td></td><td></td><td>Self-designed and 3D printed</td></tr><tr><td>Plasticine</td><td>Cernit</td><td>49655005</td><td></td></tr><tr><td>Silikonpasten, Baysilone</td><td>VWR</td><td>291-1220</td><td></td></tr><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse, C57BL/6</td><td>The Jackson Laboratory</td><td></td><td></td></tr></tbody></table>

whole-mount immunofluorescence staining, confocal imaging and 3d reconstruction of the sinoatrial and atrioventricular node in the mouse

窦房结（SAN）中起搏器细胞在生理上产生的电信号通过传导系统（包括房室结（AVN））传导，以允许整个心脏的兴奋和收缩。SAN 或 AVN 的任何功能障碍都会导致心律失常，表明它们在电生理学和心律失常发生中起根本作用。小鼠模型广泛用于心律失常研究，但SAN和AVN的具体研究仍然具有挑战性。
SAN位于终末嵴与上腔静脉的交界处，AVN位于科赫三角形的顶点，由冠状窦的孔，三尖瓣环和Todaro的肌腱形成。然而，由于体积小，传统组织学的可视化仍然具有挑战性，并且不允许在其3D环境中研究SAN和AVN。
在这里，我们描述了一种全接口免疫荧光方法，该方法允许标记的小鼠SAN和AVN的局部可视化。 全接口免疫荧光染色适用于较小的组织切片，无需手动切片。为此，解剖小鼠心脏，去除不需要的组织，然后固定，透化和阻塞。然后将SAN和AVN内传导系统的细胞用抗HCN4抗体染色。共聚焦激光扫描显微镜和图像处理可以区分淋巴结细胞和工作心肌细胞，并清楚地定位SAN和AVN。此外，还可以结合其他抗体来标记其他细胞类型，例如神经纤维。
与传统的免疫组织学相比，全卡口免疫荧光染色保留了心脏传导系统的解剖完整性，从而可以研究AVN;特别是它们的解剖结构以及与周围工作心肌和非肌细胞的相互作用。

通过使用上述协议，可以可靠地执行SAN和AVN的共聚焦显微镜成像。使用靶向HCN4的荧光抗体对传导系统进行特异性染色，并使用靶向Cx43的荧光抗体对工作心肌进行染色，可以清晰地识别完整解剖结构内的SAN（图5，视频1）和AVN（图6，视频2）。
<img alt="Figure 1"...

Watch this Scientific Journal Video about Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse at JoVE.com

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

我们为鼠心脏窦房结（SAN）和房室结（AVN）的全安装免疫荧光染色提供了分步方案。

小鼠窦房和房室结的全卡口免疫荧光染色、共聚焦成像和 3D 重建

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

whole-mount-immunofluorescence-staining-confocal-imaging-3d

Research

JoVE Journal

Medicine

5.3K 次观看.  Ludwig Maximilians University (LMU) Munich. 我们为鼠心脏窦房结（SAN）和房室结（AVN）的全安装免疫荧光染色提供了分步方案。

视频: Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

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 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 ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

光片荧光显微术在小鼠心脏研究中的应用

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

生物工程

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

高分辨率光学制图对小鼠心房电生理的评价

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

医学

用于全面心脏成像的光片显微镜和组织透明化

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

作者聚焦：用于研究啮齿动物心脏心肌结构的定制光片成像

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

光片成像揭示啮齿动物心脏的心脏结构

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

生物学

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.

鼠标窦房结的高分辨率光学测绘

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...

揭示小鼠肺静脉心肌结构

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial fibrillation (AF). PVs contain myocardial sleeves (MS) composed of cardiomyocytes. MS are implicated in the initiation and maintenance of AF, as they preserve similarities to the cardiac working myocardium, including the ability to generate ectopic electrical impulses. Rodents are widely used and may represent excellent animal models to study the pulmonary vein myocardium since cardiomyocytes are widely present all over the vessel wall. However, precise microdissection and preparation of murine PVs is challenging due to the small organ size and intricate anatomy.
We demonstrate a microscopy-guided microdissection protocol for isolating the murine left atrium (LA) together with the PVs. Immunofluorescence staining using cardiac Troponin-T (cTNT) and connexin 43 (Cx43) antibodies is performed to visualize the LA and PVs in full length. Imaging at 10x and 40x magnification provides a comprehensive view of the PV structure as well as detailed insights into the myocardial architecture, particularly highlighting the presence of connexin 43 within the MS.

作者聚焦：开发跨物种心房颤动研究的转化模型 

This protocol demonstrates microscopy-guided isolation and immunofluorescence staining of murine pulmonary veins. We prepare tissue samples containing the left atrium, pulmonary veins, and the corresponding lungs and stain them for cardiac Troponin T and Connexin 43.

小鼠肺静脉心肌套的显微解剖和免疫荧光染色

Pulmonary veins (PVs) are the major source of ectopic beats in atrial arrhythmias and play a crucial role in the development and progression of atrial ...

小鼠窦房和房室结的全卡口免疫荧光染色、共聚焦成像和 3D 重建

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此视频中的章节

在胚胎小鼠心脏采用免疫荧光心肌发展分析

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

光片荧光显微术在小鼠心脏研究中的应用

高分辨率光学制图对小鼠心房电生理的评价

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

光片成像揭示啮齿动物心脏的心脏结构

从成年小鼠的分离，培养和窦房结肌细胞的功能分析方法

鼠标窦房结的高分辨率光学测绘

小鼠肺静脉心肌套的显微解剖和免疫荧光染色