Name: visualizing lymph node structure and cellular localization using ex-vivo confocal microscopy
Uploaded: 2019-08-09T13:30:00
Description: Watch this Scientific Journal Video about Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy at JoVE.com

这项工作得到了NIH(R01 AI43458至H.L.W.)的支持。

该协议由哈佛医学院动物常设委员会和布里格姆妇女医院批准,协议2016N000230。
1. 用于实验的老鼠
<ol><li>在B6背景上使用8周大的雄性小鼠和雌性小鼠进行抗体混合。</li>
	<li>使用 CX3CR1GFP/WTCCR2RFP/WT小鼠确定是否也可以将前体全 LN 成像应用于报告小鼠,而无需施用抗体混合物,并调查是否存在单核细胞,包括抗原呈现细胞和噬菌体,及其在LN中的分布。 注:CX3CR1GF...

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成像与其他技术(包括分子生物学和高维免疫分型)的结合增强了我们在其原生环境中研究免疫细胞的能力。事实上,虽然其他方法可能需要组织消化和细胞分离(这可能导致组织完整性的丧失),但使用体内或前体成像在以地理方式调查不同细胞亚型方面具有很大优势3,16.转基因小鼠菌株的可得性迅速增加,这种菌株专门针对细胞表达不同的荧光。重要的是,报?...

淋巴结 (Lun) 是卵形器官,广泛存在于全身,具有桥接先天和自适应免疫反应的关键功能。LUN过滤淋巴,以识别外来颗粒和癌细胞,以对它们进行免疫反应1。抗原呈现细胞(ApCs),T细胞和B细胞一起产生抗原特异性抗体(体液免疫)和细胞毒性淋巴细胞(细胞免疫),以消除外来颗粒和癌细胞2。因此,了解淋巴系统中免疫细胞的动态对疫苗开发和癌症免疫治疗具有重要影响。
强大的显微镜的出现-包括新的共聚焦和超分辨率显微镜-使一个非凡的进步,在理解不同的免疫细胞群体如何在他们的原生环境中如何运行3。现在,可以使用与转基因小鼠结合探针,在特定靶点4、5的控制下表达荧光蛋白,可以成像几个同时发生的细胞亚型。事实上,高维技术,包括大规模细胞学和多参数流分析,对于扩大我们对健康和疾病中不同免疫细胞的分块和功能的知识至关重要。7.然而,为了准备这些技术的样本,组织需要消化,细胞从自然环境中分离出来,在细胞悬浮液中进行分析。为了超越这些限制,并允许生物学更好的翻译,这里提出的协议的目标是应用一个简单的方法,使用库存共聚焦显微镜图像外整个淋巴结,有利于提高速度,组织与传统的免疫荧光染色相比,结构保存和细胞存活。通过这种方法,我们能够证明缺乏β-T细胞的小鼠,一种参与宿主早期防御病原体4的T淋巴细胞的亚型,与野生型小鼠相比,已经破坏了毛囊和T细胞区。这些发现使我们能够进行一项研究,其中我们证明βT细胞在淋巴器官平衡和体液免疫反应4中起关键作用。此外,该协议为探针和抗体到达淋巴结提供了一个生理途径,因为它们在原位标记使用的报告的基础上,通过组织淋巴环流进行皮下施用并消散与抗体可视化淋巴相关结构8,9, 芽中心动力学10,11,12,和目标容易进入血流13 ,14,15.

<table>
	<tbody>
		<tr>
			<td>BV421 anti-CD4</td>
			<td>BD Horizon</td>
			<td>562891</td>
			<td>GK1.5; 0.2 mg mL-1</td>
		</tr>
		<tr>
			<td>BB515 anti-CD19</td>
			<td>BD Horizon</td>
			<td>564509</td>
			<td>1D3; 0.2 mg mL-1</td>
		</tr>
		<tr>
			<td>BB515 Rat IgG2a, &#954; Isotype Control</td>
			<td>BD Horizon</td>
			<td>564418</td>
			<td>R35-95; 0.2 mg mL-1</td>
		</tr>
		<tr>
			<td>BV421 Mouse IgG2b, K Isotype Control</td>
			<td>BD Horizon</td>
			<td>562603</td>
			<td>R35-38 0.2 mg mL-1</td>
		</tr>
		<tr>
			<td>Cellview culture dish</td>
			<td>Greiner-Bio</td>
			<td>627861</td>
			<td>35x10 mm with glass bottom</td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>BD Plastipak</td>
			<td>-</td>
			<td>Insulin U-100</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech Science Brand</td>
			<td>7557</td>
			<td>size 21 x 20 cm / 100 sheets per box</td>
		</tr>
		<tr>
			<td>Microsurgery curved forceps</td>
			<td>WEP Surgical Instruments</td>
			<td>custom made</td>
			<td>12.5 cm</td>
		</tr>
		<tr>
			<td>Microsurgery curved scissors</td>
			<td>WEP Surgical Instruments</td>
			<td>custom made</td>
			<td>11.5 cm</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>BD PrecisionGlide</td>
			<td>-</td>
			<td>30 gauge &#215; &#189; inch</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Te + A1R confocal head</td>
			<td>Nikon</td>
			<td>-</td>
			<td>loaded with main 4 laser lines (405, 488, 543 and 647 nm)</td>
		</tr>
		<tr>
			<td>PE anti-F4/80</td>
			<td>BD Pharmigen</td>
			<td>565410</td>
			<td>T45-2342; 0.2 mg mL-1</td>
		</tr>
		<tr>
			<td>PE Rat IgG2a, &#954; Isotype Control</td>
			<td>BD Pharmigen</td>
			<td>553930</td>
			<td>R35-95; 0.2 mg mL-1</td>
		</tr>
		<tr>
			<td>Zeiss LSM 710 confocal microscope</td>
			<td>Zeiss</td>
			<td>-</td>
			<td>loaded with main 4 laser lines (405, 488, 543 and 647 nm)</td>
		</tr>
	</tbody>
</table>

visualizing lymph node structure and cellular localization using ex-vivo confocal microscopy

淋巴结(LUN)是体内传播的器官,与先天免疫反应可以连接适应性免疫。事实上,LN战略性地插在淋巴血管的路径中,允许组织抗原与LN中所有常驻免疫细胞亲密接触。因此,使用外体全LN成像了解细胞组成、分布、位置和相互作用,将增进有关人体如何协调局部和全身免疫反应的知识。该协议显示了在体内给荧光标记抗体进行体内给药后,使用传统的共聚焦显微镜和库存试剂,允许非常可重复且易于执行的方法的体外成像策略。通过皮下抗体注射,可以在排空LUN中标记不同的细胞群,而不影响可能由传统免疫荧光显微镜技术破坏的组织结构。

本手稿展示了在注射荧光标记抗体以染色这些器官的特定细胞群后,在不破坏其结构的情况下去除淋巴结的技术(图1和图2).
LN细胞与BV421抗CD4和BB515抗CD19和共聚焦成像分析的免疫标记的强大组合,定义了T细胞(CD4+)和B细胞(CD19+)在铀素和波普利特LN中的定位。在两个器官中,B细胞卵泡被T细胞群包围(图3,<strong class="xfig...

Watch this Scientific Journal Video about Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy at JoVE.com

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位

该协议描述了一种在不改变器官结构的情况下在排空淋巴结中成像不同细胞群的技术。

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

这种可重复且易于执行的协议使用前维生淋巴结成像策略来跟踪体内施用荧光标记抗体对局部二级淋巴器官的排出。演示此技术允许过程的特定可视化，从而促进协议的成功执行。这是一个非常简单和直接的技术，任何人都可以执行。唯一具有挑战性的方面是鼠标处理，这需要一些培训。与传统的荧光染色方法相比，该技术具有改进速度组织作用保存和细胞生存能力的益处。培训程序将是布鲁娜·泰特松，一个从实验室的技术人员。稀释感兴趣的抗体，结合到适当的荧光团，在100微升PBS内淋巴结成像，或50微升PBS的波普莱特淋巴结成像。对于内淋巴结成像，将100微升的抗体鸡尾酒装入一毫升胰岛素注射器，配备30分半英寸针，皮下将抗体注射到实验小鼠的大腿内侧。对于波光淋巴结成像，将50微升的抗体鸡尾酒皮下注射到实验动物的一个爪垫中。然后把动物还到家里的箱子里。对于内皮排出淋巴结的收获，在注射后至少等待三个小时后，用胶带将鼠标固定在丙烯酸舞台上，并在腹部皮肤上涂抹矿物油。使用标准的显微外科剪刀和显微外科弯曲钳子，使腹部的中线皮肤切口从到双磷化过程，并分离腹部肌肉与皮肤。在垂直切口线的顶部和底部进行水平皮肤切口，在注射侧创建皮肤皮瓣，并收回皮肤以可视化淋巴结。将皮肤皮瓣固定到丙烯酸板上，并使用钳子去除半透明，通常是球形内皮排干淋巴结。对于波莱特排水淋巴结收获，注射后至少12小时，将鼠标固定在丙烯酸舞台上的易发位置。将矿物油涂抹在动物注射侧的小腿和膝盖上。接下来，在小腿从脚跟到膝盖进行中线切口，将小腿肌肉与皮肤分离。暴露流行小马。波莱特淋巴结将显示为 fossa 内的半透明球体。然后，用显微外科弯曲钳子切除淋巴结。要准备淋巴结进行成像，请将整个器官放在一个35至10毫米的培养盘中，并用钳子去除组织周围的任何脂肪。将器官放在盘子中间，用一块盐水浸泡的细腻任务雨刷器盖住组织。对于外体成像，将培养皿置于倒置共聚焦显微镜槽中，并使用共聚焦显微镜的传统光和 4-10 倍目标获得正确的对焦。从光功能更改为激光模式，并使用同位素染色、非染色或非荧光样品调整激光功率、偏移和增益，以消除自动荧光和任何未指定的染色。在 4、10 和 20X 目标下获取图像，重点是淋巴结结构和细胞分布，并使用 1024 x 1024 像素定义。然后，使用适当的成像分析软件程序来分离通道，添加感兴趣的比例和颜色，并重建 3D 视图。淋巴结细胞的免疫标记与抗CD4和抗CD19抗体和共体成像分析的强大组合允许T和B细胞在内淋巴结内的定位。在流行细胞淋巴结中，如在内结组织中，B细胞卵泡被T细胞群包围，这是淋巴结结构的标志。正如在这些图像中观察到的，噬菌体不会内化注射的抗体，表明B和T细胞染色是特异性的。此外，重新重建标签表明T和B细胞染色不重叠，进一步确认了染色的特异性。单细胞细胞在整个内结淋巴结中观察到，包括亚帽鼻窦。大多数单核细胞表示CX3XR1，其次是CCR2和CXCR31CCR2双阳性细胞。在波莱特淋巴结中观察到细胞分布和细胞表型的相同模式。两个淋巴结也表现出没有单核细胞受体表达的暗区，这些暗区被淋巴细胞占据。此外，单核细胞在淋巴结的内侧区域是稀缺的，仍然集中在组织的外侧区域，表明这些细胞主要占据淋巴结亚帽鼻窦。抗体混合物需要精确注射到淋巴系统的皮下组织，以适当排出淋巴结的抗体。该方法可应用于荧光标记药物的生物分布，以及荧光标记纳米粒子的细胞靶向研究。

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Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

A System for ex vivo Culturing of Embryonic Pancreas

A System for ex vivo Culturing of Embryonic Pancreas

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

纳米有限元分析：蛋白质定位的光激活定位显微镜和电子显微镜

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

使用荧光显微镜可视化中的细菌线虫

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Visualizing Stromule Frequency with Fluorescence Microscopy

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Cellular Redox Profiling Using High-content Microscopy

使用高含量显微镜的细胞氧化还原分析

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...