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
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 ?...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer system&#160;</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 colspan="4">In vivo instrumentation</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 colspan="4">General lab instruments</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Sunlab</td>
			<td>D-8040</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA&#160;</td>
			<td>RH basic</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette,volume 10 &#181;L, 100 &#181;L, 1000 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683884-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Microscopes</td>
		</tr>
		<tr>
			<td>Dissection stereo- zoom microscope&#160;</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 colspan="4">Software</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 colspan="4">General Lab Material</td>
		</tr>
		<tr>
			<td>0.2 &#181;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 colspan="4">Chemicals</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&#160;</td>
			<td>28908</td>
			<td>use as a 4% solution&#160;</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&#39;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 colspan="4">Drugs</td>
		</tr>
		<tr>
			<td>Fentanyl 0.5&#160;mg/10&#160;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 colspan="4">Antibodies</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG Alexa Fluor&#160;488&#160;</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 colspan="4">Other</td>
		</tr>
		<tr>
			<td>Plastic 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 colspan="4">Animals</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 ...

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