Name: retinal cryo-sections, whole-mounts, and hypotonic isolated vasculature preparations for immunohistochemical visualization of microvascular pericytes
Uploaded: 2018-10-07T14:00:00
Description: Watch this Scientific Journal Video about Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes at JoVE.

这项研究由丹麦 Lundbeck 基金会资助。

对成年雄性白化大鼠进行了优化和验证。在所有实验程序中, 动物在眼科和视力研究的 ARVO 声明中都按照《动物》的规定进行治疗。动物被二氧化碳和随后的颈椎脱位安乐死。
1. 大鼠视网膜组织制剂
<ol><li>
低温切片
	<ol>
<li>用手术刀使大鼠眼睑的后、前0.5 厘米狭缝。<ol>
<li>可选: 使用透热法燃烧器, 在嵌入时将眼睛标记在内角以统一的方式定位眼睛, 并允许垂直?...

<ol>
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D., 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=Diltiazem+reduces+retinal+neovascularization+in+a+mouse+model+of+oxygen+induced+retinopathy.">Diltiazem reduces retinal neovascularization in a mouse model of oxygen induced retinopathy.</a> Current Eye Research. 18, 20-27 (1999).</li><li>Chou, J. C., Rollins, S. D., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin+digest+protocol+to+analyze+the+retinal+vasculature+of+a+mouse+model.">Trypsin digest protocol to analyze the retinal vasculature of a mouse model.</a> Journal of Visualized Experiments. , e50489 (2013).</li><li>Hazra, S., 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=Liver+X+receptor+modulates+diabetic+retinopathy+outcome+in+a+mouse+model+of+streptozotocin-induced+diabetes.">Liver X receptor modulates diabetic retinopathy outcome in a mouse model of streptozotocin-induced diabetes.</a> Diabetes. 61, 3270-3279 (2012).</li><li>Zhang, L., Xia, H., Han, Q., Chen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+antioxidant+gene+therapy+on+the+development+of+diabetic+retinopathy+and+the+metabolic+memory+phenomenon.">Effects of antioxidant gene therapy on the development of diabetic retinopathy and the metabolic memory phenomenon.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 253, 249-259 (2015).</li><li>Dagher, Z., 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=Studies+of+rat+and+human+retinas+predict+a+role+for+the+polyol+pathway+in+human+diabetic+retinopathy.">Studies of rat and human retinas predict a role for the polyol pathway in human diabetic retinopathy.</a> Diabetes. 53, 2404-2411 (2004).</li><li>Navaratna, D., McGuire, P. 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我们提出三种视网膜制备技术, 可用于微血管性视网膜毛细血管的研究。下面, 我们提供了两种方法之间的比较, 并突出显示了协议中的关键步骤。
随着低温切片, 视网膜被切割成矢状切片, 因此, 有可能获得来自同一视网膜的大量标本。这个方法产生的数字部分使它成为抗体特异性和滴定测试的理想选择, 因为它可以防止不必要的动物牺牲。筹备工作对取得好成绩是决定性的。...

视网膜毛细血管是许多研究实验室的重点, 因为这些细胞在血管的完整性中起着重要的作用。病理条件如糖尿病视网膜病变1, 缺血2, 青光眼3有血管特征, 涉及毛细血管的功能。毛细血管见于内视网膜毛细血管丛。视网膜中央动脉提供内视网膜分支成两层毛细血管丛。内血管床位于神经节细胞和内核层之间。更深层是更加密集和复杂的, 并且在内部和外部核层数之间被本地化4,5。此外, 视网膜的某些部分还包含第三个称为径向 parapapillary 毛细血管的网络。这些是长, 直毛细血管在神经纤维之间和很少吻合互相或其他二丛6。在毛细管壁内, 毛细血管嵌在基底膜内, 线 abluminal 侧血管内皮细胞。
到目前为止, 没有这些毛细血管的唯一生物标记, 可以区分它们与其他血管细胞。血小板衍生生长因子受体β (PDGFRβ) 和神经/胶质抗原 2 (NG2) 是常用的标记, 既存在于毛细血管, 也包括其他血管细胞。毛细血管的识别由于形态学和蛋白质表达式的周细胞子集的存在而进一步复杂化.目前, 最佳的鉴定依赖于蛋白质标记的组合和周细胞在血管壁的特征定位。我们在这里演示了三种不同的组织制备技术的免疫组化 PDGFRβ/NG2 染色的大鼠视网膜微血管毛细血管,即低温切片, 全支架, 和低渗的隔离血管网络。
随着低温切片, 视网膜和巩膜被切断通过视神经。这使得神经元的所有层状结构可视化。不同的十层视网膜是明显的交换核和轴突/树突结构, 可以可视化的污点, 如苏木精/红/红或荧光核 4 ', 6-diamidino 2-苯基吲哚 (DAPI)8。代谢要求不同的层9 , 它提供了一种方法来确定厚度或总缺乏的特定层 (例如, 视网膜神经节细胞的损失是视网膜缺血10的标志之一, 11)。血管是明显的横向切开通过视网膜, 使成为可能分开地研究毛细血管丛在各自视网膜层数12,13。
传统上, 视网膜血管网的研究是在全支架上进行的。随着这种组织的准备, 视网膜被切割和扁平的花状结构。该方法是一种相对较快的组织制备技术, 能突出整体结构的视网膜血管, 因此经常被应用于小鼠视网膜新生血管的研究中。在新生小鼠和大鼠视网膜14、15、16、17、18中, 还报道了全装视网膜微血管的成功可视化19. 这些研究显示, 与新生儿视网膜14相比, 成年人的无毛细血管面积更明确的 pericytic 活动。
另一种可视化的方法是在低渗隔离后的视网膜微血管。这种组织制备技术导致视网膜血管和毛细血管被释放的神经细胞。这种类型的孤立视网膜血管网络的二维成像通常是在视网膜胰蛋白酶消化20后进行的, 用于评估糖尿病视网膜病变的血管异常, 包括周细胞丢失和毛细血管退化20,21,22。低渗隔离法提供了对视网膜血管基因和蛋白质调控反应的研究, 因为它们已经做了 rt-pcr 和西方印迹23,24,25。我们提供了一个协议, 以自由浮动的免疫组化染色的低渗隔离视网膜血管作为胰蛋白酶消化的替代品, 以检查微血管毛细血管。

<table border="0" cellpadding="0" cellspacing="0" width="902">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Geletin from porcine skin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:165px;">G2625-500G</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Albumin from chicken egg white</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:165px;">A5253-500G</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Deoxyribonuclease (DNAse) I from bovine pancreas</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:165px;">D5025-15KU</td>
			<td style="width:171px;">Dissolved in 0.15 M NaCl</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Bovine serum albumin (BSA)</td>
			<td style="width:175px;">VWR</td>
			<td style="width:165px;">0332-100G</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Normal donkey serum</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:165px;">017-000-121, lot 129348</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Rabbit anti-PDGFR&#946;</td>
			<td style="width:175px;">Santa Cruz</td>
			<td style="width:165px;">sc-432</td>
			<td style="width:171px;">1:100</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Mouse anti-NG2</td>
			<td style="width:175px;">Abcam</td>
			<td style="width:165px;">ab50009</td>
			<td style="width:171px;">1:500</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:165px;">711-585-152</td>
			<td style="width:171px;">1:100</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Fluorescein (FITC) AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:165px;">715-095-151</td>
			<td style="width:171px;">1:100</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Cy2 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:165px;">711-225-152</td>
			<td style="width:171px;">1:100</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Cy3 AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:165px;">715-165-150</td>
			<td style="width:171px;">1:100</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:165px;">D9542-1MG</td>
			<td style="width:171px;">Dissolved in DMSO</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Anti-fading mounting medium</td>
			<td style="width:175px;">Vector Laboratories</td>
			<td style="width:165px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Anti-fading mounting medium with DAPI</td>
			<td style="width:175px;">Vector Laboratories</td>
			<td style="width:165px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:392px;">Nunc Lab-Tek II 4-well chamber slide</td>
			<td style="width:175px;">Thermo Fisher Scientific</td>
			<td style="width:165px;">154526</td>
			<td></td>
		</tr>
	</tbody>
</table>

retinal cryo-sections, whole-mounts, and hypotonic isolated vasculature preparations for immunohistochemical visualization of microvascular pericytes

视网膜毛细血管在许多眼部疾病中起着重要作用。视网膜血管和微血管毛细血管的免疫组化染色技术是眼科研究的中心。选择合适的方法来可视化微血管毛细血管是至关重要的。我们用血小板衍生生长因子受体β (PDGFRβ) 和神经/胶质抗原 2 (NG2) 的抗体描述了低温切片、全支架和低渗隔离血管的视网膜微血管周细胞免疫组化染色。这使我们可以突出的优点和缺点, 三组织准备的可视化视网膜微血管毛细血管。冷冻切片提供 transsectional 可视化所有视网膜层, 但只包含少数偶尔的横向削减微血管。全芒提供了整个视网膜血管的概述, 但可视化的微循环可能是麻烦的。低渗隔离提供了一种通过去除神经细胞来可视化整个视网膜血管的方法, 但这使得组织非常脆弱。

成功的协议提供三种不同的视网膜准备可视化微血管毛细血管。每种方法都使用 PDGFRβ和 NG2 免疫联合定位和毛细血管的独特位置环绕毛细血管内皮 foridentification。
随着低温切片, 神经元层可由 DAPI 标记核的荧光密度来识别, 内、深毛细血管丛含有毛细血管, 显示 PDGFRβ和 NG2 免疫反应 (图 1)。毛细血管在血?...

Watch this Scientific Journal Video about Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes at JoVE.

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

我们展示了三种不同的组织制备技术的免疫组化显示大鼠视网膜微血管毛细血管,即低温切片, 全支架, 和低渗的隔离血管网络。

视网膜低温切片、全支架和低渗隔离血管毛细血管微血管组织免疫组化的制备

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

这些方法可以帮助回答眼科领域的关键问题，如环小体增殖或迁移。这些过程的主要优点是它们提供了各种可视化选项。演示程序的将是卡琳·德雷西格和弗兰克·布利克斯特，两个博士后在我们的实验室。为了使眼睛保持光亮，首先使用手术刀在大鼠眼睑中使后侧和前部的缝隙保持约 0.5 厘米。用钳子抓住眼睛，小心地向侧面倾斜，露出周围的组织。在结缔组织和肌肉组织中用解剖剪刀进行切口，使眼睛得到锻炼。将眼睛短暂地放在4%甲醛和磷酸盐缓冲盐水中一至两分钟。在用手术刀尖施加轻压之前，先在角膜肢上打孔，然后用钠缓冲液短暂冲洗。用解剖剪刀切割角膜肢体，用钳子切除角膜和透镜。将剩余的组织与视网膜一起吸收到PBS中4%甲醛中，两到四个小时。通过浸入索伦森含有10%蔗糖的磷酸盐缓冲液，然后吸收25%蔗糖，冷冻保护组织。一旦组织沉入容器底部，就将其转移。嵌入雅祖拉介质中，用3%的明胶从猪皮和30%白素从蛋清准备。接下来，将明胶嵌入视网膜的10微米垂直冷冻切入视神经。将冷冻片放在玻璃滑梯上，至少干燥一小时。干燥后，用 0.25%Triton-X 100 将幻灯片淹没在 PBS 中 15 分钟。然后，将PBST和1%BSA中1：100 PDGF受体β和1：500NG2原抗体的混合物滴到低温上，在4摄氏度的湿润室中过夜。第二天，将幻灯片浸入 PBST 中两次，每次 15 分钟，以清洗部分。然后滴1：100抗小鼠Alexa Fluor 594链接和1：100抗兔 FITC链接的二次抗体的混合物，用3%BSA稀释在BST中，并在黑暗中孵育一小时。孵育后，每次在PBST中冲洗玻璃滑梯15分钟。使用含有 DAPI 和盖玻片的防褪色安装介质安装染色的低温。第二种视网膜组织制备技术是全安装。切除角膜后，使用钳子的小开口运动将视网膜从视网膜色素上皮分离到视神经。用解剖剪刀释放视神经的视网膜，从视网膜外围向视神经头部开三到四条几毫米长的缝隙。将视网膜铺在玻璃滑梯上，晾干5至10分钟。将4%甲醛滴到视网膜上，固定20至30分钟。固定后，使用 PBS 冲洗。为了获得最佳效果，冲洗后直接进行免疫。首先，将PBST滴入整座，在室温下孵育15分钟。从PBST中倒出，然后加入原抗体溶液，在4摄氏度的湿润室中孵育。第二天，倒掉原抗体，滴在PBST上，每次冲洗视网膜两次，每次15分钟。浇注第二次洗涤后，将二次抗体溶液滴到视网膜上，在黑暗中室温下在湿润室中孵育一小时。在二次抗体孵育后，与以前一样用PBST洗涤两次。使用包含 DAPI 和盖滑的防褪色安装介质安装染色的整个安装。除了完全安装视网膜，视网膜血管可以通过低分位隔离来隔离。首先，将视网膜放在24井板中的一毫升去电化水中，在室温下以1.5毫米振动轨道以200 rpm转速摇动，持续一小时，然后加入200单位的DNAse 1，将视网膜血管中的落酸细胞碎片分离，在室温下再摇晃30分钟。在去层水中冲洗视网膜三次，每次五分钟，以 150 至 300 rpm 的转速摇动，以清除神经元细胞碎片。每次冲洗时，视网膜应变得更透明，表示可去除神经元细胞碎片。冲洗后，在室温下将视网膜固定为4%甲醛和PBS的1毫升10分钟。用 PBS 冲洗三次。如果继续免疫，用500微升10%驴血清在PBS中稀释1小时，在100转/小时时摇晃，阻断低分分离血管。阻断后，在600微升原抗体溶液中孵育视网膜，包括1：100PDGF受体β和1：500NG2原抗体，在10%驴血清和PBS中稀释。第二天，在PBS中冲洗视网膜网络三次，每次五分钟，然后在1：100抗小鼠Alexa Fluor 594和1：100抗兔 FITC 相关二次抗体中孵育，在 10% 驴血清和 PBS 中稀释，在室温下以 100 rpm 摇动，在黑暗中摇动一小时。在 PBST 中洗涤 5 分钟后，每毫升 DAPI 和 PBST 孵育 0.2 毫微克 15 分钟。使用 PBST 清洗三次，五分钟，同时防止光线。接下来，切割塑料巴斯德移液器的尖端。使用移液器尖端去除锋利的边缘，用 PBST 滋润巴斯德移液器，然后将视网膜血管吸入移液器并放入四孔玻璃室滑梯中。保湿步骤是重要的，以避免视网膜血管粘在巴斯德移液器内部。通过来回倾斜腔室滑动来定位视网膜血管。从腔室滑轨的井中拆下介质。液体的表面张力会将血管压平到滑轨底部。如果需要，将液体滴到视网膜血管上展开。在从室滑轨上取下塑料孔之前，确保在显微镜下正确展开。使用防褪色安装介质和盖滑来安装染色的血管。NG2免疫组织化学揭示了视网膜内侧阳性的血管。箭头表示容器中的圆形和马蹄形免疫反应。箭头指出了一个纵向切割的容器。PDGF受体β免疫组织化学显示与NG2相似的免疫反应。再次箭头和箭头指示容器中的免疫反应。此图显示了绿色 NG2 的合并，红色中的 PDGF 受体测试版和蓝色 DAPI 的合并。通过叠加三种不同的滤波器，发现NG2和PDGF受体β是共同本地化的。此图显示了一个免疫染色全安装制剂与NG2染色为红色。免疫反应性沿着表面血管网络可见，在肺膜内有强烈的染色。神经元细胞在NG2染色微血管之间以DAPI蓝色核可见。此图显示了低分位分离大鼠视网膜血管网络染色的NG2在绿色和PDGF受体β在红色。整个网络显示NG2免疫反应。PDGF受体β免疫反应在细胞生长膜中发现。在较高的放大率下，很明显PDGF受体β染色只在血管网络中的细胞索马斯中看到，表明具有抗性免疫反应。将 DAPI 染色与 NG2 和 PDGF 受体 beta 一起显示此高放大图像中两个未定义的容器壁细胞中的三个周数。按照这些程序，其他免疫染色，如平滑肌肉细胞或内皮染色可用于回答其他问题。例如，视网膜血管中的细胞在缺血之后看起来如何？

Research

JoVE Journal

Biology

Thin Sectioning of Slice Preparations for Immunohistochemistry

薄切片进行免疫组化切片的筹备工作

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

科研

生物学

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

使用激光光镊操纵隔离神经细胞在体外

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

精子发生的细胞学分析：Live和固定制剂<em&gt;果蝇</em&gt;睾丸

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

运动性的时空制图<em&gt;离体</em肠子&gt;准备工作

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

眼部微血管血管生成前维沃胆球状芽分析

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...