Name: using en face immunofluorescence staining to observe vascular endothelial cells directly
Uploaded: 2019-08-20T12:30:00
Description: Watch this Scientific Journal Video about Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly at JoVE.com

这项研究得到了国家自然科学基金（授权号81670451，81770430）、上海新星计划（授权号17QA1403000）和上海市政府科学技术委员会（授权号81670451）的支持。14441903002, 15411963700).

所有动物实验均按照上海交通大学动物资源委员会批准的实验规程进行。
1. 灌注鼠标主塔
<ol><li>简单地说，用五巴比妥钠（50毫克/千克体重）的腹内注射麻醉12周大的C57BL/6小鼠。轻轻捏紧尾巴，确认正确的麻醉。 注：如果未观察到任何运动，动物被充分麻醉以开始实验。</li>
	<li>用胶带将鼠标的爪子粘在一叠纸巾上。</li>
	<li>用钳子抓住老鼠的皮肤，用剪刀把皮肤从腹部?...

<ol>
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内皮液暴露于许多原生因子，包括脂质、炎症中介和流体剪切应激1，11，12。直接观察原位内皮细胞为分析细胞形态、细胞间结和分子表达模式的变化，以响应损伤刺激具有特殊优势。
以前的研究提供了两种不同的面免疫性化学技术来观察动脉壁4，5的内皮。其?...

血管的早期变化主要在内皮内展开，内皮球在血液和血管壁之间具有选择性屏障，其细胞间紧密结复合物1。大量证据表明，血流在调节内皮功能2中的机械作用起着关键作用。流体剪切应力，一种由血流产生的摩擦力，根据不同血管位点2、3的特定流动模式，不同形状内皮细胞形态和功能。与稳定流动（s-flow）区域（如动脉的直线段）相比，动脉粥样硬化病变优先发生在血流紊乱（d-flow）部位，如血管曲率、流量分频和分支点。因此，直接观察内皮形态和分子表达模式，应有助于深入了解不同流动范式下内皮细胞的结构和功能表型。
培养的内皮细胞可能无法像在体内那样表达实际表型，部分原因是流体剪切应力、周围细胞因子和细胞细胞细胞外基质相互作用的影响。为了达到这个目的，可以使用传统的免疫组织化学在横截面研究完整的内皮细胞单层。然而，内皮单层是如此薄和脆弱，它通常无法清楚地观察到。En 面部免疫组织化学已用于观察内皮内表面，但结果要么复杂或不稳定，因为内皮容易从底层组织剥离，或者只是大鼠或兔子动脉壁的一部分，其壁厚，安装4，5。
老鼠模型在许多方面比其他动物有相当大的优势。在这里，我们采用一种经过修饰的面内免疫荧光技术来分析C57BL/6小鼠的主动脉和胸主动脉的内皮细胞。这种技术已被广泛用于研究不同流动模式的内皮病理生理学，以及动脉粥样硬化6、7、8、9、10的发展。这种方法使科学家能够清楚地观察内皮的整个表面，并比较不同流体剪切应力下区域中特定蛋白质的表达模式。

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		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Antifade mountant</td>
			<td style="width:126px;">Servicebio</td>
			<td style="width:134px;">G1401</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Delicate Forceps</td>
			<td style="width:126px;">RWD Life Science</td>
			<td style="width:134px;">F11001-11</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Delicate Scissors</td>
			<td style="width:126px;">RWD Life Science</td>
			<td style="width:134px;">S12003-09</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Dissecting Forceps</td>
			<td style="width:126px;">RWD Life Science</td>
			<td style="width:134px;">F12005-10</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Mciro Spring Scissors</td>
			<td style="width:126px;">RWD Life Science</td>
			<td style="width:134px;">S11001-08</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Polyoxyethylene octyl phenyl ether (Triton X-100)</td>
			<td style="width:126px;">Amresco</td>
			<td style="width:134px;">M143</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Polysorbate 20 (Tween 20)</td>
			<td style="width:126px;">Amresco</td>
			<td style="width:134px;">0777</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">VCAM-1 antibody</td>
			<td style="width:126px;">Abcam</td>
			<td style="width:134px;">ab134047</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">VE-Cadherin antibody</td>
			<td style="width:126px;">BD Biosciences</td>
			<td style="width:134px;">555289</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Alexa Fluor 555 labeled anti-rabbit IgG</td>
			<td style="width:126px;">invitrogen</td>
			<td style="width:134px;">A-31572</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Alexa Fluor 488 labeled anti-rat IgG</td>
			<td style="width:126px;">invitrogen</td>
			<td style="width:134px;">A-21208</td>
			<td style="width:174px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laser Scanning Microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td style="width:134px;"></td>
			<td style="width:174px;"></td>
		</tr>
	</tbody>
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using en face immunofluorescence staining to observe vascular endothelial cells directly

内皮表型和形态的异常变化被认为是动脉粥样硬化发病机制的初始事件。直接观察完整的内皮，为了解功能失调的内皮细胞的细胞和分子事件提供有价值的信息。在这里，我们描述了一种经过修饰的面免疫荧光染色技术，使科学家能够获得完整的内皮表面的清晰图像，并原位分析分子表达模式。该方法简单可靠，用于观察主塔不同部位的整个内皮单层。这项技术可能是了解动脉粥样硬化病理生理学的有前途的工具，特别是在早期阶段。

一只12周大的C57BL/6小鼠被安乐死，并注入含有40单位/mL肝素的正常盐水，然后预冷冻4%的甲醛。小鼠主塔在解剖显微镜下暴露（图1），解剖，纵向切开（图2）。血管内皮细胞的En面免疫荧光染色，如图3和表1所示。血管细胞粘附蛋白-1（VCAM-1）表达的En面免疫荧光，以VE-卡塞林作为内皮标记物，...

Watch this Scientific Journal Video about Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly at JoVE.com

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

在这里，我们提出了免疫荧光染色方案，以直接观察小鼠主尾的内皮细胞。该技术在研究不同流动模式的内皮细胞细胞和分子表型以及动脉粥样硬化的发展时非常有用。

使用 En 面免疫荧光染色直接观察血管内皮细胞

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of ...

该协议帮助人们分析内皮细胞形态，以及不同流体剪切应力下区域分子的表达。该技术的主要优点是，它提供了不同流体剪切应力下的血管内皮细胞的直接评估。执行此过程时，关键步骤是使用新鲜准备的甲醛，并执行良好的灌注。从 12 周前的 C57BL/6 鼠标麻醉开始此过程，如文本协议中所述。通过轻轻捏住尾巴来确认正确的麻醉。用胶带把鼠标的爪子用胶带把鼠标的爪子用一叠纸巾粘起来。用钳子握住老鼠的皮肤，用一把剪刀从腹部切到胸部顶部。用一把锋利的剪刀打开肋骨下方的腹腔。用钳子抬起胸骨，然后切割隔膜。然后切开肋骨，露出胸腔。用剪刀切断肝脏上方的维娜卡瓦。压力通过左心室，使动脉树承受五分钟，预冷却的正常盐水每毫升肝素含有40个单位，直到肺部和肝脏变得苍白。然后在PBS中用预冷却的4%甲醛进行4分钟。去除所有的肌肉，器官和脂肪，直到大母骨暴露。将鼠标放在解剖显微镜下。使用细腻的钳子和一把精致的剪刀在解剖显微镜下清楚地暴露大动脉，并尽可能干净地去除大动脉的结缔组织。用一把细腻的剪刀将胸主动脉从心脏解剖到腹腔躯干。将大母板放入六厘米的细胞培养盘中，并配以PBS。纵向切开主动脉，首先沿着较小的曲线，然后使用微切口切开主动脉拱的三根分支，包括主动脉、左普通胡萝卜和沿大曲线的左亚克拉维安动脉，直到出现直线段。用1%聚氧乙烯八基苯醚在PBS中渗透大母10分钟。然后在室温下用10%正常山羊血清在含有2.5%多糖酸20的三分液盐水中阻断大母，在12孔细胞培养板中阻断1小时。接下来孵育大母油在阻断缓冲液中含有5克每毫升兔子抗VCAM1，和五克每毫升大鼠抗V-Cadherin，在4摄氏度过夜。用洗涤溶液冲洗样品三次后，在室温下应用荧光结合二次抗体一小时，保持在黑暗的地方。在洗涤液中再冲洗三次。用 DAPI 对大道进行对，对着 10 分钟，保持在黑暗的地方。然后在洗涤液中冲洗染色大干母板三次。将大母板放在带流明表面的盖滑上，然后缓慢地移动到以前掉在盖滑上的防淡安装溶液。轻轻伸展大母，使试样保持平整。反转盖滑，放在玻璃滑梯上。注意避免试样和玻璃之间有任何气泡。使用激光扫描共体显微镜检查大母体。使用 Image-Pro Plus 软件分析不同通道在 en 面图像中所需区域的颜色强度。显示小鼠大动脉血管内皮细胞的免疫荧光染色图像。血管细胞粘附蛋白一的免疫荧光，以及以维-卡德林作为内皮标记的表达，在小鼠大动脉不同区域的不同流动模式下显示。DAPI 也反面显示细胞核，以便更好地可视化。在显微镜下查看 Z 堆栈时，内皮和平滑的肌肉细胞可以很容易地通过细胞核形态来区分。内皮细胞核为椭圆形，比主轴形状、平滑的肌肉细胞核大。与稳定流动下的区域相比，VCAM-1在扰流区域、大弓曲率较小的区域、大弓拱门曲率较大的区域和胸大大项的表达更为丰富。在这里，小鼠大血管曲率较小的区域表示为 d 流区域，曲率和胸角较大。通过正确的修改，该技术还可用于血管渗透性分析，以及大分子在内皮细胞内膜的位置。

Research

JoVE Journal

Biology

Isolation of Valvular Endothelial Cells

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

科研

生物学

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

直接进入成年斑马鱼的肾细胞移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

米色/布里特细胞的间质血管细胞的分离和分化

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Robust 3D DNA FISH Using Directly Labeled Probes

强大的3D直接标记的探针DNA鱼

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Isolation of Murine Valve Endothelial Cells

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

一氧化氮和超氧化物歧化酶恩的人脸检测在完整的动脉内皮细胞层

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

一个简单的生物测定血管内皮生长因子的评价

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

En Face Preparation of Mouse Blood Vessels

En Face Preparation of Mouse Blood Vessels

面对小鼠血液制备

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...