Name: en face preparation of mouse blood vessels
Uploaded: 2017-05-19T13:00:00
Description: Watch this Scientific Journal Video about En Face Preparation of Mouse Blood Vessels at JoVE.com

作者的研究活动得到了国家卫生研究院对Abe博士（HL-130193，HL-123346，HL-118462，HL-108551）的资助。

小鼠部分颈动脉结扎和分离小鼠主动脉和颈动脉用于面部免疫染色的方案由机构动物护理和使用委员会（IBT 2014-9231）批准。 左侧部分颈动脉结扎<ol><li>通过在桌子上放置一个12英寸x 14英寸的加热垫来准备手术空间，并用大的干净的手术罩盖住垫子和桌面。调整吊架的臂，使立体显微镜的视野位于加热垫的中心区域。 </li><li> 打开桌面上的加热垫，并将3设置控制拨盘设?...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+arterial+blood+pressure+in+different+strains+of+mice.">Comparison of arterial blood pressure in different strains of mice.</a> Am J Hypertens. 14 (5), 405-408 (2001).</li><li>Jinguji, Y., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fiber+dependent+axial+organization+of+fibronectin+fibrils+in+the+basal+lamina+of+the+chick+and+mesenteric+artery.">Stress fiber dependent axial organization of fibronectin fibrils in the basal lamina of the chick and mesenteric artery.</a> Endothelium. 2, 35-47 (1994).</li><li>Neckel, P. H., Mattheus, U., Hirt, B., Lust, L., Mack, A. 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当处理小鼠血管时，重要的是记住内皮是脆弱的，并且任何过量的机械力将损伤内皮细胞。例如，如果容器被强力灌注，则血管壁破裂或分离，当使用手动注射器灌注脉管系统时，这可能容易发生。 为了获得恒定的灌注压力，我们使用120厘米水柱压力的重力灌注系统。据报道，不同于应变的小鼠的平均动脉压在130和170厘米H 2 O 14之间 。因此，我们使...

通过光学显微镜进行组织病理学研究，常规处理生物组织三维片段进行石蜡包埋，然后进行切片和染色。石蜡包埋的组织样品可以在所有三维中为几毫米。然而，为了光学显微镜的目的，它必须首先切片，使得光可以通过然后染色，使得薄部分产生足够的成像对比度。由于切片样品的厚度通常为5-10微米，所以每次只能看到整个样品的二分之一。可以收集连续的部分，并且在单独成像每个部分之后，对3D图像进行计算机辅助重建，但是这是一个繁琐的工作。血管的组织病理学，特别是研究动脉粥样硬化的发病机制，具有独特的问题。动脉粥样硬化是一种发展的重点疾病局部发生血流紊乱的地区。此外，该疾病是在内膜内开始的，一个由大量动脉的内皮细胞和细胞外基质的单层组成的薄组织。由于这些原因，使用分段血管来定位和研究早期病变是一个挑战，因为可以容易地错过切片病变。即使部分确实包括患病区域，在介质和外膜中只能看到含有内皮细胞和其他血管壁细胞的5-10μm部分。 整个安装面（发音为änfäs）的准备工作使我们可以从主动脉根部一直到髂总动脉，调查血管表面的广泛区域，如整个主动脉。使用这样的标本用特异性抗体和其他特异性探针染色，可以确定病变的位置，同时也可以在内皮细胞中发生各种分子事件动脉粥样硬化形成，如蛋白质的表达，定位和翻译后修饰的变化。除了研究动脉粥样化形成外，在面部制剂中观察到的内皮细胞形状被用作区域时间平均血流模式的指标。这些数据对于研究原位内皮细胞的机械信号是很重要的。为此，常规的组织学横切血管是无用的。因此，对于血管医学和生物学，特别重要的是获得一种用于制造血管的面部制备技术，其允许观察血管表面的广泛区域以及血管的较深的地下区域。 正如Jelev和Surchev 1所述 ，血管生物学家已经开发了各种观察血管内衬的方法。一些巧妙的方法是在20世纪40年代和50年代开发的。他们使用这些方法<html>能够研究排列血管内表面的内皮细胞的基本组织。然而，由于这些准备工作的准备方法（所谓的Hautchen方法2,3,4或容器表面5的剥离）以及样品染色的方式，并不总是可以获得不间断的形态信息从血管表面进入血管壁的较深的区域。全面安装和面部血管制剂联合免疫荧光染色使我们不仅可以研究这些细胞内皮细胞的形态和蛋白质表达和定位，而且还将这些研究扩展到血管壁的内皮下区域。早期研究使用血管，面部免疫荧光染色剂的制剂在20世纪80年代开始出现6 ，随着激光扫描共聚焦显微镜和最近的多光子显微镜的出现，现在可以获得免疫荧光染色的面容器样品中的血管壁结构的清晰的对焦图像以及活体动物中的血管网络这些基于计算机的成像技术创建了对焦光学分割图像，并且通过堆叠这样的图像，可以获得组织中血管壁和血管网络的重建3D图像，另外，一个可以生成沿着重建图像12,13的Z轴形成的部分的图像。 在本文中，我们将说明一种制备小鼠主动脉和颈动脉免疫荧光染色的方法。面对准备即使在这些容器被实验操作之后也可以制造。例如，可以部分地连接颈动脉，然后在这样的手术之后进行面部制备。因此，我们还将在本文中描述我们如何在颈动脉上进行部分结扎。与大型动物如大鼠，兔子和人类进行类似的制备相比，小鼠血管的尺寸较小且更易碎，因此需要对外科手术分离血管进行处理以及进行抗体染色和显微镜检查。因为最常用的遗传修饰动物模型是老鼠，因此许多研究者在不伤害它们的情况下处理小鼠血管变得至关重要。在本手稿中，我们将介绍如何在小鼠主动脉和颈动脉进行面部准备时处理小鼠血管。为了展示的目的，我们将使用野生型C57 / b6小鼠。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride injection solution USP 500ml bag</td>
			<td>Fisher Scientific</td>
			<td>NC9788429</td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Fisher Scientific</td>
			<td>12556005</td>
		</tr>
		<tr>
			<td>6-0 coated vicryl suture</td>
			<td>Ethicon</td>
			<td>J833G</td>
		</tr>
		<tr>
			<td>AF488 goat anti-rat IgG&#160;</td>
			<td>Life Technologies</td>
			<td>A11006</td>
		</tr>
		<tr>
			<td>AF546 goat anti-rabbit IgG&#160;</td>
			<td>Life Technologies</td>
			<td>A11035</td>
		</tr>
		<tr>
			<td>Anti-CD144 (Ve-Cad)</td>
			<td>BD Biosciences</td>
			<td>BD555289</td>
		</tr>
		<tr>
			<td>Anti-VCAM-1 (H-276) Rabbit polyclonal IgG</td>
			<td>Santa Cruze Biotechnology</td>
			<td>Sc-8304</td>
		</tr>
		<tr>
			<td>Aoto Flow System</td>
			<td>Braintree Scientific</td>
			<td>EZ-AF9000</td>
		</tr>
		<tr>
			<td>Autoclave Wrap. 24x24in</td>
			<td>Cardinal Health</td>
			<td>4024</td>
		</tr>
		<tr>
			<td>Blunt retractors, 2.5mm wide</td>
			<td>Fine Science Tools</td>
			<td>18200-10</td>
		</tr>
		<tr>
			<td>Caprofen (Rimadyl)&#160;</td>
			<td>zoetis</td>
			<td>NADA#141-199</td>
		</tr>
		<tr>
			<td>Chlorhexidine Scrub, 2%</td>
			<td>Med-Vet International</td>
			<td>RXCHLOR2-PC</td>
		</tr>
		<tr>
			<td>Curity gauze sponges 2x2</td>
			<td>Cardinal Health</td>
			<td>KC2146</td>
		</tr>
		<tr>
			<td>Electric heating pad, 12X14</td>
			<td>Fisher Scientific</td>
			<td>NC0667724</td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11152-10</td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-11</td>
		</tr>
		<tr>
			<td>Micro cover glass 22x50mm</td>
			<td>VWR</td>
			<td>48393059</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-18</td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Equitech-Bio</td>
			<td>GS05</td>
		</tr>
		<tr>
			<td>Paraformaldehyde Solution 4% in PBS</td>
			<td>Santa Cruze Biotechnology</td>
			<td>SC-281692</td>
		</tr>
		<tr>
			<td>Petri Dishes 100x15mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade mountant with DAPI</td>
			<td>Life Technologies</td>
			<td>P-36935</td>
		</tr>
		<tr>
			<td>Puritan cortton swabs</td>
			<td>VWR</td>
			<td>10806-005</td>
		</tr>
		<tr>
			<td>Puritan Mini cotton tipped aplicators</td>
			<td>VWR</td>
			<td>82004-050</td>
		</tr>
		<tr>
			<td>Round handled Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12076-12</td>
		</tr>
		<tr>
			<td>Silk Suture 6/0</td>
			<td>Fine Science Tools</td>
			<td>18020-60</td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>ROBOZ</td>
			<td>RS-5601</td>
		</tr>
		<tr>
			<td>Strabismus Scissors</td>
			<td>Fine Science Tools</td>
			<td>14075-09</td>
		</tr>
		<tr>
			<td>Super Grip Forceps</td>
			<td>Fine Science Tools</td>
			<td>00649-11</td>
		</tr>
		<tr>
			<td>Transparent Dressing</td>
			<td>Cardinal Health</td>
			<td>TD-26C</td>
		</tr>
		<tr>
			<td>Triton X-1000</td>
			<td>Fisher Scientific</td>
			<td>AC327371000</td>
		</tr>
	</tbody>
</table>

en face preparation of mouse blood vessels

石蜡包埋组织的切片常规用于研究组织组织学和组织病理学。然而，很难从这些部分确定三维组织形态。此外，检查的组织切片可能不包含组织内为进行研究目的所必需的区域。后一种限制阻碍血管的组织病理学研究，因为血管病变以聚焦方式发展。这需要一种使我们能够从其表面到更深的区域调查血管壁的广泛区域的方法。一个完整的血管准备满足了这一要求。在本文中，我们将展示如何使小鼠主动脉和颈动脉进行面部准备，并对共聚焦显微镜和其他类型的荧光成像进行免疫荧光染色。

图3中显示了内皮的典型的面部免疫荧光图像。该图像显示了在肋间动脉（大暗蛋形区域）的开口附近获取的小鼠主动脉的单个光学部分。主动脉用抗VE-钙粘蛋白（绿色）和抗VCAM-1（血管细胞粘附分子-1）（红色）进行双重染色。每个内皮细胞在粘附连接处用绿色线性染色来概括。由于样本的微小不均匀，一些粘附点在该光学部分之外。在已知发生扰动血流的?...

Watch this Scientific Journal Video about En Face Preparation of Mouse Blood Vessels at JoVE.com

En Face Preparation of Mouse Blood Vessels

描述了制备小鼠颈动脉和主动脉的方法。当用特异性抗体进行免疫荧光染色时，这样的制剂使得我们能够研究蛋白质的定位和通过共聚焦显微镜在整个血管壁内鉴定细胞类型。

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 ...

Research

JoVE Journal

Biology

En Face Preparation of Mouse Blood Vessels

Preparation of Dissociated Mouse Cortical Neuron Cultures

游离的小鼠皮层神经元文化的制备

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

科研

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

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 ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

内源性的免疫荧光分析及外源着丝粒着丝粒蛋白

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

小鼠精减数分裂染色体的制备

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...