我们感谢克里斯蒂娜 Pyrgaki, 陶塘, 和艾莉森北从 Bioimaging 资源中心在洛克菲勒大学的援助和支持。我们也感谢 Xiphias 的电影编辑。这项工作得到了人类前沿科学计划组织 (PC) 的支持。

根据洛克菲勒大学机构动物护理和使用委员会批准的程序, 对动物进行了护理和试验。
1. 组织准备
<ol><li>用20毫升1x 磷酸盐缓冲盐水 (PBS) 在4摄氏度内进行标准心内灌注, 直到血液完全从组织中取出。</li>
	<li>将灌流液转换为20毫升的固定液 (4% 多聚甲醛 (粉煤灰) 在 1x PBS) 4 摄氏度, 直到颈部和尾部有明显的僵硬。 注意: 粉煤灰是有毒的。避免接触皮肤、眼睛和粘膜。解决方案应在通风罩内进行。</li>
	<li>仔细解剖脂肪垫的兴趣, 以消除整个脂肪垫, 而不损害它。使用钝端钳避免捏或挤压组织。避免用任何皮毛污染解剖组织。</li>
	<li>将4% 只粉煤灰中的组织固定在 1x PBS 中过夜4摄氏度。对于每个脂肪垫, 后修复与〜10毫升固定溶液在15毫升圆锥管。</li>
	<li>用 1x PBS 在室温下 (RT) 冲洗组织3次 (每1小时)。</li>
	<li>将组织的短期 (长达2周) 存储在 1x PBS, 4 摄氏度, 并保护免受光照。对于长期存储 (例如, 1-2 年), 用0.05% 叠氮化钠 (作为防腐剂) 将缓冲器转换为 1x PBS。 注意: 叠氮化钠在口服或通过皮肤吸收时具有剧毒。浓缩的叠氮化钠溶液 (5% 或更高) 应在通风罩下处理。</li>
</ol>2. Delipidation 和通透
时间: 1-2 天
<ol><li>用 B1n 缓冲器 (表 1) (v/v) 准备20%、40%、60% 和80% 甲醇梯度,例如, 用于 20% methanol/B1n 缓冲器, 混合20毫升甲醇和80毫升 B1n 缓冲器。将所有缓冲区存储在摄氏4摄氏度。甘氨酸晶体将沉淀从60% 和 80% methanol/B1n 缓冲由于饱和。避免去除晶体;使用液体溶液进行以下洗涤。 注意: 甲醇是挥发性的、刺激性的和易燃的。避免皮肤或眼睛接触。</li>
	<li>在解剖显微镜下轻轻地去除样品中的毛发。任何附着在样品上的毛发、皮棉或杂物都会在成像过程中产生阴影。把被清洗的样品转移到新的管子里。</li>
	<li>在冰上或摄氏4摄氏度执行以下步骤, 除非另有说明。对于小样本 (例如, 幼瘦小鼠的后皮下/perigonadal 脂肪垫), 使用2毫升离心管和1.6 毫升的溶液。对于大的样品或高脂含量的样品 (例如, 肥胖小鼠的脂肪垫), 使用5毫升管和4毫升的溶液。<ol>
<li>将含有样品的管子水平放置在 100 rpm 的轨道振动筛上。确保样品能在管子内自由移动。</li>
	</ol>
</li>
	<li>在指定时间 (表 100%) 的20%、40%、60%、80% 和 2 methanol/B1n 缓冲区的分级系列中脱水样品。最小化结转解决方案。</li>
	<li>Delipidate 样品与100% 二氯甲烷 (DCM) (3 倍), 每一个孵化时间显示在表 2。样品应该下沉到管子的底部在第二和第三 DCM 洗涤的末端。如果没有, 延长潜伏期, 以确保充分 delipidation (例如, 从30分钟延长到1-2 小时)。 注意: DCM 应在通风罩内处理。使用玻璃容器储存和离心管, 是由聚丙烯为孵化。离心管不应用于 DCM 的长期贮存。由聚苯乙烯制成的培养皿和血清吸管与 DCM 不相容。DCM 是高度波动的。快速将样品转化为新鲜溶液, 防止脱水。</li>
	<li>清洗两次与100% 甲醇为指定的时间 (表 2)。</li>
	<li>可选: 如果样品不充分灌注, 血红蛋白的颜色从剩余的红细胞可能导致强的自发荧光。漂白剂与5% 小时2O2在甲醇 (1 容量 30% H2O 2容量甲醇) 隔夜在5°c。</li>
	<li>水化反向 methanol/B1n 缓冲区系列中的示例: 80%、60%、40%、20% methanol/B1n 和 100% B1n 缓冲区 (2 倍), 表示时间 (表 2)。</li>
	<li>用 PTxwH 缓冲器 (表 1) 在 RT 上清洗样品2小时。</li>
	<li>将 delipidated 示例存储在 4° C (最多1年) 的 PTxwH 缓冲区中, 或立即进行染色步骤。</li>
</ol>3. 全芒染色
时间: 8-10 天
注: 除另有说明外, 所有下列步骤均应在 RT 中进行, 并可进行震动和保护。对于小样本, 使用2毫升离心管与1.6 毫升的溶液。对于大样本, 使用5毫升管与4毫升的溶液。建议首先验证组织或甲醇处理组织切片的小块上的抗体。
<ol><li>将 PTxwH 缓冲中的主要抗体稀释为推荐浓度。离心稀释抗体溶液在〜 2万 x g 10 分钟, 以防止引入抗体沉淀或聚合。</li>
	<li>用主抗体溶液在指定的时间内孵化 delipidated 样品 (表 2)。</li>
	<li>用 PTxwH 缓冲液在一系列孵化步骤中冲洗样品: 5 分钟, 10 分钟, 15 分钟, 20 分钟, 1 小时, 2 小时, 4 小时, 和过夜。</li>
	<li>稀释 PTxwH 缓冲中的二次抗体以推荐浓度。离心稀释抗体溶液在〜 2万 x g 10 分钟, 以防止引入抗体沉淀或聚合。 注意: 为了避免组织自发荧光引起的高背景, 推荐在红色和远红色区域显影的继发抗体。根据显微镜上可用激光线的数量, 在一个样本中可以同时成像多达3-4 个标记。</li>
	<li>用第二抗体溶液在所述时间内孵化样品 (表 2)。</li>
	<li>洗涤样品与 PTxwH 缓冲在一系列孵化步骤: 5 分钟, 10 分钟, 15 分钟, 20 分钟, 1 小时, 2 小时, 4 小时, 和过夜。</li>
	<li>可选: 对于易碎的组织类型, 在4摄氏度的 1x PBS 中, 用4% 只粉煤灰固定染色组织, 以帮助保持组织形态学。 注意: 此步骤可能会增加成像背景。没有必要对脂肪组织执行这一步骤。</li>
	<li>在一系列孵化步骤中, 用 1x PBS 冲洗样品: 5 分钟, 10 分钟, 30 分钟。</li>
	<li>在解剖显微镜下轻轻地去除样品中的皮棉。</li>
</ol>4. 组织清除
时间: 1-2 天
<ol><li>可选: 在琼脂糖中嵌入样品, 以方便样品安装, 用于光学显微镜。 注意: 这个步骤强烈建议脂肪组织在成像过程中稳定其形状。<ol>
<li>用1% 琼脂糖在 1x PBS (w/v) 上制备嵌入溶液。冷却嵌入溶液为 ~ 40 °c, 以避免暴露样品过热。</li>
		<li>将清洗过的样品放入模具中 (例如, 培养皿或称重船), 并将其排列到所需位置。避免任何液体的结转。将嵌入的解决方案轻轻地倒在样本上。避免气泡。</li>
		<li>让琼脂糖在 RT 中完全凝固. 切出包含样本的块。 注意: 包括夜间孵化在内的所有步骤都应在 RT 中进行, 并能震动和保护光线。对于小样本, 使用2毫升离心管与1.6 毫升的溶液。对于大样本, 使用5毫升管与4毫升的溶液。</li>
	</ol>
</li>
	<li>用 H2O: 25%、50%、75% 和 100% (3 倍) 在甲醇梯度中脱水样品 (表 2)。 注: 样品可以在100% 甲醇步骤过夜。</li>
	<li>在 100% DCM 中孵育样本1小时, 晃动 (3 次)。样品应下沉到底部的管在每个 DCM 洗涤结束。如果没有, 延长孵化时间, 以确保完全去除甲醇。示例可以在 100% DCM 步骤中过夜。</li>
	<li>用轻度震动在二苄醚 (DBE) 中孵化样品, 实现折射率匹配。 注: 样品最终会在可见光下变得透明。 警告: DBE 是危险的。避免与皮肤接触。用双层手套在通风罩内处理。一旦它被 DBE 污染, 就扔掉手套。使用玻璃容器储存和离心管 (聚丙烯) 的孵化。离心管不应用于长期贮存 DBE。由聚苯乙烯制成的培养皿和血清吸管与 DBE 不相容。用100% 乙醇清洗任何泄漏。</li>
	<li>用新鲜的 DBE 对样品进行孵育, 2 小时进行轻度晃动. 将样品保存在深色的 RT 中, 或者直接进行成像。 注: 建议样品在一个月内进行成像。虽然某些显影比其他的更稳定, 但不推荐在这个阶段长期储存样品。</li>
</ol>5. 显微学
<ol><li>与 DBE 兼容的光片显微镜成像。 注意: 只有通过有机溶剂为基础的成像和与 DBE 的折射率相匹配的目标才可以作为 DBE 浸没成像的浸渍目标。<ol>
<li>将琼脂糖嵌入的样品装入一个能防止成像过程中运动的支架上。将样品浸入一个 DBE 的腔内。</li>
		<li>收集一个 z 栈覆盖整个样本 (如, 1-2X 放大), 以获得整个组织分布/结构的兴趣标记。</li>
		<li>切换到具有更高放大倍数 (例如, 4-12X 放大) 的目标, 放大图像详细结构所感兴趣的区域。 注: 胶原蛋白是脂肪组织细胞外基质的主要贡献者, 它围绕着所有的脂肪细胞13。胶原蛋白有一个典型的发射光谱, 范围约400毫微米到 550 nm14。成像样品与488激光线 (绿色) 和收集发射光在波长范围内, 位于胶原的发射光谱 (如, 525-550 nm) 将提供组织自发荧光信号, 这是以前显示, 以描绘脂肪细胞轮廓和整体组织结构12。</li>
	</ol>
</li>
	<li>用倒置共焦或双光子显微镜成像。<ol>
<li>将琼脂糖嵌入的样品放入一个玻璃底部的腔内滑动, 不接触任何塑料部件 (或其他可密封的 DBE 的成像室)。确保 DBE 不泄漏。</li>
		<li>选择更长的工作距离的目标, 以达到更深的渗透。 注: 成像应采用倒置共焦或双光子显微镜通过玻璃底部的会议厅幻灯片。避免在样品和浸渍目标之间的直接接触, 这是没有建议的 DBE 成像。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

Adipo 是一种简单和稳健的方法来清除脂肪组织, 这可以很容易地执行在常规实验室设置。与其他溶剂型清除方法 (如 iDISCO/iDISCO +10、11、12、Adipo) 相比, 对于清除脂肪组织和脂肪含量高的其他组织尤为优化。delipidation 的步骤完全消除脂肪, 从而促进 immunolabeling 整个组织, 并大大减少光散射, 允许端到端成像, 而不丢失任何 XY 分辨?...

直到最近, 脂肪组织被认为是一种非晶的脂肪细胞集合体。在过去的几十年里, 我们的理解变得更加复杂, 现在人们认识到脂肪是一种复杂的器官, 含有不同类型的脂肪细胞, 以及脂肪体的前体细胞、免疫单元、成纤维细胞、血管和神经投射。这些脂肪细胞间的相互作用对脂肪组织和生物体生理和病理生理学有显著的影响1。虽然新的研究已经解开了某些相互作用的重要分子机制, 但更全面的理解需要对三维 (3D) 的整个组织进行可靠的结构分析。
我们目前对脂肪组织形态学的认识主要基于薄切片 (5 微米) 的组织学分析和相对高放大率成像 (超过 10X)2,3。但是, 这种方法有几个重要的局限性。首先, 复杂的丝状结构, 如交感神经和血管, 被称为在脂肪功能4,5,6,7的重要作用, 很难评估通过薄的部分。第二, 由于其表面看似无定形, 缺乏代表性的结构单元, 因此很难欣赏仅基于切片染色的脂肪组织结构。第三, 脂肪组织具有很高的脂质含量, 在获得一致的连续切片方面产生了挑战, 适合于3D 解剖重建, 这是一种用于研究全脑形态学8的常规方法。考虑到这些因素, 很大的需要一个整体安装的方法, 可以提供3D 可视化整个脂肪库, 而仍然实现细胞分辨率。
3D. 由于光散射的遮蔽作用, 整个器官的容积成像具有挑战性。生物组织中光散射的主要来源来自脂质水界面。虽然在过去一个世纪里, 消除油脂分散的努力一直在进行, 但最近有许多创新9。一种新开发的组织清除方法是 immunolabeling 3D 成像的溶剂清除器官 (iDISCO/iDISCO +)10,11。然而, 脂肪组织提出了一个特殊的挑战, 鉴于其高水平的血脂, 因此, 需要额外的修改 iDISCO/iDISCO + 协议, 以充分提取脂质, 同时保护组织免于崩溃。我们开发的修改后的协议, 现在称为 Adipo, 采用甲醇/二氯甲烷为基础的脂肪组织 delipidation, 以达到最佳的透明度, 适合高分辨率的容积成像12。由于 delipidation 步骤主要止渴内在表达荧光蛋白, 如 GFP 和 RFP, 这种蛋白质的可视化必须通过 immunolabeling 来实现。总的来说, 这一简单而健壮的协议可以应用于研究脂肪细胞的组织水平组织、脂肪祖细胞的谱系追踪以及发育过程中的脂肪形态发生。

<table><tbody><tr><td>1x phosphate buffered saline</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148-1KG</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412SK-4</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100-500ML</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P2287-500ML</td><td></td></tr><tr><td>Heparin</td><td>Sigma Aldrich</td><td>H3393-100KU</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>270991</td><td></td></tr><tr><td>Hydrogen peroxide 30%</td><td>Fisher Scientific</td><td>325-100</td><td></td></tr><tr><td>Benzyl ether</td><td>Sigma Aldrich</td><td>108014</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500500</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>71289-5G</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-1</td><td></td></tr><tr><td>Rabbit polyclonal anti-Tyrosine Hydroxylase</td><td>Millipore</td><td>AB152</td><td>1:200 dilution</td></tr><tr><td>Goat polyclonal anti-CD31/PECAM-1</td><td>R&amp;amp;D Systems</td><td>AF3628</td><td>Final concentration of 2 &amp;micro;g/mL</td></tr><tr><td>Rat monoclonal anti-CD68, Clone FA-11</td><td>Bio-Rad</td><td>MCA1957</td><td>Final concentration of 2 &amp;micro;g/mL</td></tr><tr><td>Donkey anti-rabbit IgG (H+L) Alexa Fluor 647</td><td>Jackson ImmunoResearch</td><td>711-605-152</td><td>Final concentration of 5-10 &amp;micro;g/mL</td></tr><tr><td>Donkey anti-goat IgG (H+L) Alexa Fluor 568</td><td>Invitrogen</td><td>A11077</td><td>Final concentration of 5-10 &amp;micro;g/mL</td></tr><tr><td>Donkey anti-rat IgG (H+L) Alexa Fluor 647</td><td>Jackson ImmunoResearch</td><td>712-605-153</td><td>Final concentration of 5-10 &amp;micro;g/mL</td></tr><tr><td>Imaging chamber</td><td>ibidi</td><td>80287</td><td></td></tr><tr><td>Light sheet microscope</td><td>LaVision BioTec</td><td>Ultramicroscope II</td><td></td></tr><tr><td>Imaging software</td><td>LaVision BioTec</td><td>Imspector software</td><td></td></tr><tr><td>Microscopy visualization software</td><td>Bitplane</td><td>Imaris</td><td></td></tr></tbody></table>

adipo-clear: a tissue clearing method for three-dimensional imaging of adipose tissue

脂肪组织在能量稳态和体温中起着核心作用。它由不同类型的脂肪细胞, 以及脂肪细胞的前体, 免疫单元, 纤维化, 血管和神经投射组成。虽然细胞类型规范的分子控制和这些细胞的相互作用已经越来越多地被划定, 通过可视化它们的分布和结构, 可以更全面地了解这些脂肪驻留细胞整个组织。现有的免疫组化和免疫荧光方法分析脂肪组织学依赖薄石蜡嵌入切片。然而, 薄部分只捕获一小部分组织;结果, 这些结论可能会被分析的组织部分所偏向。因此, 我们开发了一种脂肪组织清除技术, Adipo 明确, 以允许全面的三维可视化的分子和细胞模式的整个脂肪组织。Adipo 是从 iDISCO/iDISCO +, 并作出了具体的修改, 以彻底去除储存在组织中的脂质, 同时保持本机组织形态学。结合光片荧光显微镜, 我们在这里展示了使用 Adipo 的方法来获得高分辨率的整个脂肪组织的体积图像。

Adipo-清晰的准备完整的脂肪垫可以在3D 成像, 以分析如何在瘦身和肥胖状态下的组织形态学和细胞相互作用的影响。该方法可以很容易地应用于在绿色通道中采集组织自发荧光信号来分析一般脂肪结构。我们以前已经表明, 脂肪覆盖物的自发荧光信号与 perilipin 染色有利, 一个常用的标记, 以概述成熟脂肪细胞12。例如, 使用低放大率 (1.3X) 的光片显微镜扫描...

Watch this Scientific Journal Video about Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue at JoVE.com

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

由于脂质含量高, 脂肪组织在使用传统的组织学方法进行可视化方面具有挑战性。Adipo 是一种组织清除技术, 它允许强健的标记和高分辨率的脂肪组织的荧光成像。在这里, 我们描述的方法, 样品制备, 预处理, 染色, 清除和安装的成像。

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 ...

adipo-clear-tissue-clearing-method-for-three-dimensional-imaging

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

27.0K 次观看.  The Rockefeller University. 由于脂质含量高, 脂肪组织在使用传统的组织学方法进行可视化方面具有挑战性。Adipo 是一种组织清除技术, 它允许强健的标记和高分辨率的脂肪组织的荧光成像。在这里, 我们描述的方法, 样品制备, 预处理, 染色, 清除和安装的成像。

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

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

成像在细胞水平上通过3DISCO清除完整生物系统

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

科研

神经科学

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

本地化识别，切除脂肪小鼠油库，并

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

医学

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

脂肪组织中的血管和神经纤维的共染色

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

免疫与感染

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

免疫标记肾组织的光学清除与成像

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

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

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

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 ...

生物学

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

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 ...

<meta charset="utf8"></head><body>人 SATMVEC：分离和脂肪细胞串扰研究

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

<meta charset="utf8"></head><body>作者聚焦：通过皮下脂肪组织微血管研究洞察心脏代谢疾病

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

Three-Dimensional Adipocyte Culture as a Model to Study Cachexia-Induced White Adipose Tissue Remodeling

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic wasting has been proposed to be stimulated by several inflammatory mediators, which may disrupt the integrative physiology of adipose tissues and other tissues such as the brain and muscle. In this scenario, the tumor can survive at the host&#39;s expense. In recent clinical research, the intensity of depletion of the different fat deposits has been negatively correlated with the patient&#39;s survival outcome. Studies have also shown that various metabolic disorders can alter white adipose tissue (WAT) remodeling, especially in the early stages of cachexia development. WAT dysfunction resulting from tissue remodeling is a contributor to overall cachexia, with the main modifications in WAT consisting of morpho-functional changes, increased adipocyte lipolysis, accumulation of immune cells, reduction of adipogenesis, changes in progenitor cell population, and the increase of &#34;niches&#34; containing beige/brite cells.
To study the various facets of cachexia-induced WAT remodeling, particularly the changes&#160;progenitor cells and beige remodeling, two-dimensional (2D) culture has been the first option for in vitro studies. However, this approach does not adequately summarize WAT complexity. Improved assays for the reconstruction of functional AT ex vivo&#160;help the comprehension of physiological interactions between the distinct cell populations. This protocol describes an efficient three-dimensional (3D) printing tissue culture system based on magnetic nanoparticles. The protocol is optimized for investigating WAT remodeling induced by cachexia induced factors (CIFs). The results show that a 3D culture is an appropriate tool for studying WAT modeling ex vivo and may be useful for functional screens to identify bioactive molecules for individual adipose cell populations applications and aid the discovery of WAT-based cell anticachectic therapy.

This protocol describes a three-dimensional (3D) magnetic printing culture system that permits dissection of white adipose tissue (WAT) remodeling induced by a conditioned medium from cancer cells. Using a 3D culture system of UCP1+ adipocytes that express a green fluorescent protein (GFP) allows the study of beige adipocytes contributing to adipose tissue remodeling.

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic ...

Adipo: 三维脂肪组织成像的组织清除方法

摘要

探索更多视频

此视频中的章节

成像在细胞水平上通过3DISCO清除完整生物系统

本地化识别，切除脂肪小鼠油库，并

脂肪组织中的血管和神经纤维的共染色

免疫标记肾组织的光学清除与成像

三维白色脂肪组织结构的全装染色可视化

通过甲基异硫清除和三D成像探索脂肪组织结构

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

Three-Dimensional Adipocyte Culture as a Model to Study Cachexia-Induced White Adipose Tissue Remodeling