Name: exploring adipose tissue structure by methylsalicylate clearing and 3d imaging
Uploaded: 2020-08-19T15:00:00
Description: Watch this Scientific Journal Video about Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging at JoVE.com

这项工作得到了 INSERM 的支持， 蔚蓝海岸大学，以及法国国家研究机构（ANR）通过未来实验室信号投资（ANR-11-LABX-0028-01）的资助，通过第2学院"Systémes复合体"和学院4"复杂与多样性"项目UCA JEDI（ANR-15-IDEX-01） Médical基金会（+quipe FRM DEQ20180839587）和J.G.青年调查员计划（ANR18-CE14-0035-01-GILLERON）。我们还感谢由阿尔卑斯海洋局和Région PACA委员会资助的C3M成像核心设施，该设施也得到IBISA显微镜和成像平台Céte d'Azur（MICA）的支持。我们感谢马里恩·杜索在组织准备方面提供的技术帮助。我们感谢艾比·卡特里斯，UCA国际科学能见度，为手稿的校对阅读。

该协议已经过测试，并验证了所有小鼠和人类白色脂肪组织库。人类和小鼠脂肪组织是根据欧洲法律收集的，并经法国和瑞典伦理委员会批准。
1. 小鼠和人类白色脂肪组织的固定
<ol><li>将收获的小鼠或人类白色脂肪组织浸入至少 10 mL 的 PBS 中，其中含有 4% 的甲醛 （PFA） 在 15 mL 塑料管中。</li>
	<li>在室温下在滚动板上摇动塑料管 1 小时。</li>
	<li>将塑料管在 4°C 下放在滚动?...

<ol>
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脂肪组织内在病理进展过程中发生的改变，如肥胖，对于理解病理背后的机制至关重要。揭示脂肪组织中这种机制的开创性研究基于全球方法，如全脂肪组织蛋白质组学21、流细胞学,22、23和转录组学24、25。,2522此外，还利用组织,学分析26、27、28、29，,<sup c...

肥胖症的特点是脂肪组织质量增加，并已成为一个重大的全球公共卫生问题，因为肥胖者患心血管疾病、2型糖尿病、肝病和一些癌症的风险增加。
脂肪组织的基本生理功能是调节全身葡萄糖和脂质平衡1，2。1,在喂养期间，脂肪细胞（即脂肪组织的主要细胞）将膳食提供的葡萄糖和脂质的过量储存到甘油三酯中。在禁食过程中，脂肪细胞将甘油三酯分解成非酯化脂肪酸和甘油，以维持身体的能量需求。在肥胖症的发育过程中，脂肪组织通过增加脂肪细胞的大小（肥大）和/或（增生）数量来扩大脂肪细胞1，以增加其储存能力。当脂肪组织的膨胀达到其极限时，患者中一个恒定的极易变量，剩余的脂质会积聚到其他代谢器官，包括肌肉和肝脏3、4，4导致其功能衰竭，并引发肥胖相关的心代谢并发症1,1，5。因此，确定控制脂肪组织扩张的机制是临床的关键挑战。
肥胖期间在脂肪组织内记录的形态修饰与其病理功能障碍有关。一些染色程序已经被用来描述脂肪组织的组织，包括行为素6，血管标记7，脂滴标记8，和特定的免疫细胞标记9,9，10。然而，由于脂肪细胞（50至200μm）的巨大直径11，因此必须从三维分析整个组织的很大一部分，以便准确分析肥胖期间观察到的戏剧性的结构 AT 变化。然而，由于光线不穿透不透明组织，因此不可能使用荧光显微镜在大型组织样本中进行3D成像。组织清除方法，使其透明已经报告在文献（审查，见12），允许一个人清除组织，并执行深入，整个组织荧光显微镜。这些方法为评估健康和患病组织中的 3D 细胞组织提供了前所未有的机会。每种描述的方法都有优点和缺点，因此需要根据所研究的组织仔细选择（如要回顾，见13）。事实上，有些方法需要长时间的潜伏期和/或使用昂贵,、有毒或难以获得的材料或化合物，例如14、15、16、17、18、19。,1516,17,18,19利用一个世纪前沃纳·斯帕尔特霍尔茨（Werner Spalteholz）用于清除组织20的第一批化合物之一，我们建立了一个用户友好且价格低廉的协议，非常适用于清除任何实验室中所有小鼠和人类脂肪组织库，这些实验室具有典型的设备，包括化学罩、温度控制轨道摇床和共生显微镜。

<table>
	<tbody>
		<tr>
			<td>1.5 mL microtubes</td>
			<td>Eppendorff tubes - Dutscher</td>
			<td>33528</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL plastic tubes</td>
			<td>Falcon tubes - Dutscher</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm round glass coverslip</td>
			<td>Mariendfeld</td>
			<td>0117580</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL glass bottle</td>
			<td>Wheaton</td>
			<td>986546</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse-alexa647-conjugated antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-605-150</td>
			<td>Dilution: 1/100</td>
		</tr>
		<tr>
			<td>anti-rabbit-alexa647-conjugated antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-605-152</td>
			<td>Dilution: 1/100</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-aldrich</td>
			<td>A6003</td>
			<td></td>
		</tr>
		<tr>
			<td>CD301-PE antibody</td>
			<td>Biolegend</td>
			<td>BLE145703</td>
			<td>Dilution: 1/100</td>
		</tr>
		<tr>
			<td>CD31 antibody</td>
			<td>AbCam</td>
			<td>ab215912</td>
			<td>Dilution: 1/50</td>
		</tr>
		<tr>
			<td>Commercial 3D analysis software - IMARIS</td>
			<td>Oxford instrument</td>
			<td></td>
			<td>with Cell module</td>
		</tr>
		<tr>
			<td>Confocal microscope - Nikon A1R</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi</td>
			<td>ThermoFisher</td>
			<td>D1306</td>
			<td>Stock Concentration: 5 mg/mL; dilution 1/1000</td>
		</tr>
		<tr>
			<td>Deoxycholate</td>
			<td>Sigma-aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Glut4 antibody</td>
			<td>Santa Cruz</td>
			<td>sc-53566</td>
			<td>Dilution: 1/50</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Lectin-DyLight649</td>
			<td>Vector Lab</td>
			<td>DL-1178-1</td>
			<td>Stock Concentration : 2 &#181;g/&#181;L; IV Injection: 50 &#181;L/mice</td>
		</tr>
		<tr>
			<td>Metallic imaging chamber equipped with glass bottom - AttoFluor Chamber</td>
			<td>Thermofisher</td>
			<td>A7816</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl salicylate</td>
			<td>Sigma-aldrich</td>
			<td>M6752</td>
			<td></td>
		</tr>
		<tr>
			<td>Perilipin antibody</td>
			<td>Progen</td>
			<td>651156</td>
			<td>Dilution: 1/50</td>
		</tr>
		<tr>
			<td>Phalloidin-alexa488</td>
			<td>ThermoFisher</td>
			<td>A12379</td>
			<td>Dilution: 1/100</td>
		</tr>
		<tr>
			<td>TCR-&#946;-PB antibody</td>
			<td>Biolegend</td>
			<td>BLE109225</td>
			<td>Dilution: 1/100</td>
		</tr>
		<tr>
			<td>TH antibody</td>
			<td>AbCam</td>
			<td>ab112</td>
			<td>Dilution: 1/50</td>
		</tr>
		<tr>
			<td>Triton X100</td>
			<td>Sigma-aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-aldrich</td>
			<td>P416</td>
			<td></td>
		</tr>
	</tbody>
</table>

exploring adipose tissue structure by methylsalicylate clearing and 3d imaging

肥胖是一个主要的全球公共卫生问题，增加了患心血管疾病、2型糖尿病和肝病的风险。肥胖症的特点是脂肪组织 （AT） 质量增加，由于脂肪细胞增生和/或肥大，导致其三维结构的深刻重塑。事实上，在肥胖期间，AT的最大扩张能力对于肥胖相关病理学的发展至关重要。这种 AT 膨胀是一种重要的恒向机制，能够适应过多的能量摄入，并避免有害脂质溢出到其他代谢器官，如肌肉和肝脏。因此，了解导致 AT 扩展失败的结构重塑是临床适用性高的根本问题。在这篇文章中，我们描述了一种简单而快速的清除方法，在我们的实验室中经常使用这种方法，通过荧光成像来探索小鼠和人类白色脂肪组织的形态。这种优化的AT清除方法在任何配备化学罩、温度控制轨道摇床和荧光显微镜的标准实验室中都很容易实现。此外，使用的化合物是现成的。重要的是，此方法允许人们通过染色各种标记来专门可视化腺细胞、神经元和血管网络以及先天和自适应免疫细胞分布来解析 3D AT 结构。

使用此处描述和图1 中总结的程序，我们能够分别对图2A 和图 2B中所示的人类和小鼠白色脂肪组织进行污渍和光学清除。清除的组织被转移到金属成像室进行共体成像（图3A）。清除极大地改善了我们能够获取的组织图像的深度（图3B和图3C）。使用 4 倍目标（参?...

Watch this Scientific Journal Video about Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging at JoVE.com

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

在这里，我们描述了一种简单、廉价和快速的清除方法，通过荧光成像，使用标记的组合来可视化血管、核、免疫细胞、神经元和脂滴涂层蛋白，从而解析小鼠和人类白色脂肪组织的 3D 结构。

通过甲基异硫清除和三D成像探索脂肪组织结构

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

这些协议允许分析非常大的人类在小鼠脂肪组织芭蕾显微镜上的三维结构。促进病理功能障碍与脂肪组织结构变化的联系。与其他明确的协议相比，这种清除过程非常简单，非常适用于在温暖的小鼠脂肪组织中清除人类，并使用毒性较低的溶剂，如乙醇或甲基水杨酸盐。首先将收获的小鼠或人类白色脂肪组织浸入PBS中至少10毫升4%的功率甲醛中，在化学罩下优先地将15毫升的塑料管浸在PBS中。在室温下摇动卷板上的组织一小时，然后将管子在四摄氏度下将管子转移到滚动板上过夜。第二天早上，在室温下将固定的白色脂肪组织冲洗在10毫升PBS中5分钟，去除所有固定剂的痕迹，将组织转移到PBS中含有10毫升0.3%甘氨酸的15毫升管中，在室温下以每分钟100转的速度在室温下孵育1小时。当剩余的自由醛组被淬火后，将组织浸入含有10毫升0.2%Triton X-100的15毫升管中，在37摄氏度下摇晃两小时。在孵化结束时，用10毫升0.2%Triton X-100和20%DMSO治疗组织，在37摄氏度下一夜之间摇晃。第二天早上，将组织浸入含有10毫升0.1%Tween 20、0.1%Triton X-100、0.1%脱氧二甘酸酯和20%DMSO的15毫升塑料管中，在37摄氏度下至少孵育24小时，并摇动。在孵化结束时，在轨道摇床上以每分钟100转100转的速度用10毫升0.2%Triton X-100的PBS冲洗组织，一小时。随后在 37 摄氏度下浸入 15 毫升 0.2%Triton X-100、10% DMSO 和 3%BSA 中 12 小时，在 37 摄氏度下摇晃。用于染色白色脂肪组织。将组织转移到 PBS 中 0.2%Triton X-100、10%DMSO 和 3%BSA 的 300 微升中，辅以 1.5 毫升微管中感兴趣的主要抗体，用铝箔保护其免受光线的感染，将管放入 50 毫升猎鹰管中。将管子放入轨道摇床中，在37摄氏度的高温下孵育两天。在孵化结束时，在0.2%Triton X-100、10%DMSO和3%BSA的10毫升中，在PBS中冲洗组织两次，在37摄氏度下冲洗5小时，并防震防光。第二次冲洗后，用10毫升新鲜0.2%Triton X-100洗涤组织， 10%DMSO 和 3%BSA 和 PBS 在 37 摄氏度下进行 16 至 48 小时摇晃，然后将组织转移到含有 300 微升的 1.5 毫升管中，其中含有 0.2%Triton X-100、10%DMSO 和 3%BSA，辅以适当的二级抗体，并使用铝箔保护管免受光。在摇床上37摄氏度下孵育两天后，在0.2%Triton X-100、10%DMSO和3%BSA的10毫升中清洗组织，在37摄氏度下在37摄氏度下用震动防止光线。第二次洗涤后，将组织冲洗成含有10毫升新鲜0.2%Triton X-100、10%DMSO和3%BSA的管，在37摄氏度下在37摄氏度下16至48小时。接着是两小时在10毫升PBS中洗涤，温度为37摄氏度，有震动和光线保护。用于在PBS洗涤结束时清除的白色脂肪组织，将上升发送乙醇浓度序列的组织和10毫升浸入，在室温下每浓度孵育两小时，并如所示，震动不受光线影响。第二天早上，将组织浸入一个20毫升的玻璃瓶中，瓶盖塑料瓶中含有5毫升甲基水杨酸盐。在这个阶段，白色脂肪组织仍然清晰可见。摇动容器，在室温下防止光线至少两小时。白色脂肪组织完全移植。用于染色和清除白色脂肪组织的 3D 共生成像安装一个金属成像室，该成像室配有玻璃底部，并在烟罩中将组织传输到腔室。用新鲜的甲基水杨酸盐填充腔室。通过添加一个小盖滑来稳定组织和一个大盖滑来关闭腔室，完成腔室的安装。将腔室置于倒置共生显微镜阶段，以低放大倍率进行组织成像，生成整个脂肪组织样本的几立方厘米 3D 贴图。生成 3D 地图后，使用 20 倍长距离空气目标，在分辨率和深度之间提供良好的比，以更高的放大率选择多个区域进行组织采样。对于单元格分段，将 3D 堆栈转换为软件格式以释放内存空间。打开软件的单元模块之前。将细胞检测设置更改为等离子膜染色，并选择用于描绘细胞外围标记的荧光通道。然后设置阈值，提供预期单元格大小的范围并运行分段。用肉眼观察清除对人与小鼠白脂肪组织的影响。清除也大大提高了能够获取的组织的图像深度。便于整个组织 3D 成像和 4 倍放大的 3D 地图生成。3D 映射还允许在 20 倍放大到 2 毫米的深度时选择不同的感兴趣区域。脂质液滴和脂肪细胞的特异性染色可分别使用佩里平和抗胶原4抗体实现。血管可以在牺牲前不久使用CD31抗体或静脉注射Lectin DyLight 649来检测。使用抗CD301植物红素抗体和抗TCRβ太平洋蓝色抗体可以可视化巨噬细胞和T细胞。使用抗酪氨酸羟基酶抗体可检测外周神经网络。使用这些标记可以确定脂肪细胞和脂肪组织内的血管网络密度的均值和大小分布。遵循职业时间至关重要，因为样品制备不良不会产生良好的图像质量。该协议可以与先进的成像系统相结合，也可以与后处理图像分析免费软件相结合。

Research

JoVE Journal

Biology

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

科研

生物学

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

ACT-PRESTO：生物组织清算和体积成像方法免疫标记

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

三维白色脂肪组织结构的全装染色可视化

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Optical Clearing of Plant Tissues for Fluorescence Imaging

用于荧光成像的植物组织的光学清除

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator (Video) | JoVE 

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...