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 分辨率。除了光片荧光显微镜, 它提供了快速扫描大组织, 共焦和双光子显微镜也可以用来获得更大的分辨率和更多的细节。
基于甲醇/DCM 的 delipidation 是一个关键步骤。delipidation 不足会导致图像模糊, 尤其是对组织的核心。重要的是要确保充分去除脂质通过观察脂肪样本下沉在 DCM。含有组织类型的混合物 (例如, 附睾和睾丸附加的附睾脂肪) 的样本可能不会完全下沉, 即使扩展 DCM 孵化。然而, 在 DCM 中孵化这些样本应该达到完整的 delipidation。由于这些有机溶剂中蛋白质的变性, 某些抗体可能与 delipidation 步骤不相容。因此, 选择合适的抗体成为这个协议的另一个关键步骤。建议首先验证甲醇处理过的组织切片或 Adipo 处理的小块组织中的抗体。同样, 化学染料与甲醇/DCM/DBE 的相容性也应在应用前进行测试。
该协议的一个局限性是内在表达荧光蛋白的淬火。有机溶剂基 delipidation 和清除步骤主要变性这种蛋白质。为了可视化内源荧光蛋白, 需要使用抗体标记。适当的抗体组合的可用性限制了执行高复用成像的能力。目前可用的光片显微镜只允许我们想象多达4个通道。
Adipo 是特别有用的可视化丝状结构和细胞种群的密度相对较低的脂肪组织。然而, 成像密集信号变得有限的这种方法。当抗体被用来标记致密信号时, 它们可以被位于样品表面的表位所隔离, 阻止进入组织内部。因此, 建议使用小型化学探针对致密结构进行染色。由于同样的问题, Adipo 在褐色脂肪组织中的应用是有限的。褐色脂肪是一种结构致密的脂肪库。除了密集的血管和神经支配, 它还紧密地挤满了小脂肪细胞, 其中含有大量的线粒体17。当应用 CD31 和 TH 染色与上文所述的褐色脂肪相同的过程, 只有样品表面被标记 (数据没有显示)。此外, 棕色脂肪产生强烈的组织 autofluoresence, 导致低信噪比在成像过程中。建议将褐色脂肪切成小块, 并在可能时使用化学探针。
总体而言, Adipo 可以同时在整个脂肪组织中对多个感兴趣的结构进行高分辨率的分析。使用这种方法, 你可以分析神经投射、血管、免疫细胞和脂肪体等结构如何在整个脂肪垫中相互作用。它通过避免切片或选择感兴趣的区域提供无偏见的成像数据。Adipo 也可用于研究脂肪的发育, 如早期形态发生的事件, 以及脂肪细胞祖和前体细胞在世系追踪研究中的分布。除了脂肪外, Adipo 还可以促进3D 全端分析其他组织的高脂含量或周围脂肪, 如乳腺和脂肪淋巴结。该方法也为研究人体脂肪组织在生理和病理条件下的组织学提供了机会。

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&#38;D Systems</td>
			<td>AF3628</td>
			<td>Final concentration of 2 &#181;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 &#181;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 &#181;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 &#181;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 &#181;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) 的光片显微镜扫描后皮下脂肪垫 (psWAT) 显示脂肪细胞的小叶组织 (图 1A和B)。更详细的信息, 如脂肪细胞的大小, 可以通过放大放大 (4X) 感兴趣的区域 (图 1C和D) 来显示。
Adipo 是特别有用的可视化丝状结构, 如神经投射和血管, 这是挑战捕捉或跟踪薄部分。交感神经系统 (SNS) 在控制脂肪和发热脂肪组织4,5中起着至关重要的作用。成像一个 psWAT 垫染色的酪氨酸羟化酶 (TH), 一个标记为 SNS, 揭示了结构, 显示为大神经丛, 血管支配, 以及密集的终端树枝状在组织实质 (图 2A-H)。此外, 日 + 实质预测显示 psWAT 内的区域变化, 腹股沟部分具有较高的密度相对于 dorsolumbar 部分 (图 2E-H;辅助影片 1)。我们以前的研究表明, SNS 终端树枝状可以用 Imaris 软件的 FilamentTracer 工具进行计算跟踪和重建, 以评估神经支配12的密度。
已知脂肪组织有严重的血管化。脂肪代谢需求的变化通常与其血管6、7的动态重塑有关。对全组织血管进行健壮快速的分析可以为血管重塑提供额外的无偏分析。利用血小板内皮细胞黏附分子 (PECAM-1, 又称 CD31) 作为标记血管的标志物, 我们观察到所有脂肪体都与整个组织中的毛细血管接触 (图 3A-H;补充电影 2), 支持高需求的有效养分和氧交换脂肪。
免疫细胞是脂肪组织的另一个重要组成部分。在肥胖状态下, 脂肪组织会发炎, 伴随着炎症巨噬细胞的浸润, 形成 "冠状" 结构, 围绕死脂肪体15,16。肥胖动物的脂肪垫由于体积大、脂肪含量高, 特别难以清除。然而, Adipo 的扩展版本 (在表 2中描述的大组织或高脂肪组织) 可以达到一致清除整个高脂肪的组织。例如, 用16周高脂肪饮食喂养的老鼠的附睾脂肪显示了致密的 "冠状结构", immunolabeled 由 CD68, 贯穿整个组织 (图 4A和B)。重要的是, 从不同位置拍摄的光学部分在整个组织深度 (~ 4-5 毫米) 显示同样尖锐的图像, 表明组织的完全清除 (图 4C-F)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58271/58271fig1.jpg"> 图 1: 利用自体荧光信号分析脂肪组织形态学.所有面板都是光片荧光显微镜 (LSFM) 图像的 Adipo 清晰的准备 psWAT 垫隔离从一个8周大的 C57Bl/6J 雄性老鼠在 RT。通过使用绿色通道扫描清除的样本来收集自体荧光信号。dorsolumbar 区域 ( A)和腹股沟区域(B)的光学剖面 (从样品中间的剖面) 1.3X 目标。(C、 D)来自A和B的盒装区域的高放大倍数 (4X) 光学部分。淋巴结由星号指示。刻度条在每个面板中都显示。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58271/58271fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58271/58271fig2.jpg"> 图 2: 3D 脂肪组织中交感神经支配的影像学.所有的面板是 LSFM 图像的 psWAT 标记酪氨酸羟化酶 (TH) (与图 1相同的样本)。1.3X 目标所采取的重建 dorsolumbar 区(A)和腹股沟区(B)的最大预测。(C、 D)从A和B中间的光学部分。(E、F)来自C和D的盒装区域的高放大倍数 (4X) 光学部分。(G、H)在 TH (绿色) 和自体荧光 (洋红) 之间叠加的高放大光学部分。箭头表示交感神经支配的不同模式: (1) 神经丛;(2) 血管神经支配;(3) 实质树枝状。淋巴结由星号指示。刻度条在每个面板中都显示。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58271/58271fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58271/58271fig3.jpg"> 图 3: 3D 脂肪组织血管成像.所有面板都是标记为 psWAT 的 CD31 (与图 1中相同的示例) 的 LSFM 图像。(A、B)1.3X 目标所采取的重建 dorsolumbar 区(A)和腹股沟区(B)的最大预测。(C、D)从A和B中间的光学部分。(E、F)来自C和D的盒装区域的高放大倍数 (4X) 光学部分。(G、H)CD31 (红色) 和自体荧光 (青色) 之间叠加的高放大光学部分。淋巴结由星号指示。刻度条在每个面板中都显示。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58271/58271fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58271/58271fig4.jpg"> 图 4: 3D 成像的 "冠状结构" 在脂肪组织.所有的面板是 LSFM 的图像 Adipo 清晰的准备 eWAT 垫隔离从雄性老鼠喂养高脂肪饮食16周。样品与 CD31 和 CD68 immunolabeled。(A、B)被重建的样品的最大投射以总深度超过4毫米(a) x-y 视图。(B) Y Z 视图。(C)光学部分从被表明的深度在B。刻度条在每个面板中都显示。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58271/58271fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>缓冲区</td>
			<td>化学</td>
			<td>最终浓度</td>
		</tr>
<tr>
<td>B1n 缓冲区</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>甘 氨 酸</td>
			<td>0.3 米</td>
		</tr>
<tr>
<td></td>
			<td>海卫 X-100</td>
			<td>0.1% (v/v)</td>
		</tr>
<tr>
<td></td>
			<td>H2O</td>
			<td>溶剂</td>
		</tr>
<tr>
<td></td>
			<td>叠氮化钠 (防腐剂, 可选)</td>
			<td>0.01% (w/v)</td>
		</tr>
<tr>
<td></td>
			<td>用氢氧化钠调节 pH 值为7</td>
			<td></td>
		</tr>
<tr>
<td>PTxwH 缓冲区</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>10x PBS</td>
			<td>1x</td>
		</tr>
<tr>
<td></td>
			<td>海卫 X-100</td>
			<td>0.1% (v/v)</td>
		</tr>
<tr>
<td></td>
			<td>吐温20</td>
			<td>0.05% (v/v)</td>
		</tr>
<tr>
<td></td>
			<td>肝 素</td>
			<td>2µg/毫升</td>
		</tr>
<tr>
<td></td>
			<td>H2O</td>
			<td>溶剂</td>
		</tr>
<tr>
<td></td>
			<td>叠氮化钠 (防腐剂, 可选)</td>
			<td>0.01% (w/v)</td>
		</tr>
</tbody></table>表 1: 缓冲区和解决方案列表.此表包含 Adipo 中使用的缓冲区的配方。为了长期储存缓冲器, 建议将叠氮化钠作为防腐剂加入。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>缓冲区</td>
			<td>小组织</td>
			<td>大组织 (或高脂肪含量)</td>
			<td>温度</td>
		</tr>
<tr>
<td>20% methanol/B1n 缓冲器</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>40% methanol/B1n 缓冲器</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>60% methanol/B1n 缓冲器</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>80% methanol/B1n 缓冲器</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>100% 甲醇</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>Dcm</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>Dcm</td>
			<td>1小时</td>
			<td>2-3 小时或一夜之间</td>
			<td>4°C</td>
		</tr>
<tr>
<td>Dcm</td>
			<td>30分钟</td>
			<td>2小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>100% 甲醇</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>100% 甲醇</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>可选: 5% 小时2O2/甲醇</td>
			<td>一 夜 之间</td>
			<td>一 夜 之间</td>
			<td>4°C</td>
		</tr>
<tr>
<td>80% methanol/B1n 缓冲器</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>60% methanol/B1n 缓冲器</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>40% methanol/B1n 缓冲器</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>20% methanol/B1n 缓冲器</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>4°C</td>
		</tr>
<tr>
<td>B1n 缓冲区</td>
			<td>30分钟</td>
			<td>1小时</td>
			<td>Rt</td>
		</tr>
<tr>
<td>B1n 缓冲区</td>
			<td>一 夜 之间</td>
			<td>一 夜 之间</td>
			<td>Rt</td>
		</tr>
<tr>
<td>PTxwH 缓冲区</td>
			<td>2小时</td>
			<td>2小时</td>
			<td>Rt</td>
		</tr>
<tr>
<td>PTxwH 缓冲区</td>
			<td>存储</td>
			<td>存储</td>
			<td>4°C</td>
		</tr>
<tr>
<td>原发性抗体孵化</td>
			<td>3天</td>
			<td>4-5 天</td>
			<td>Rt</td>
		</tr>
<tr>
<td>次生抗体孵化</td>
			<td>3天</td>
			<td>4-5 天</td>
			<td>Rt</td>
		</tr>
</tbody></table>表 2: delipidation 和染色的孵化时间.此表包含协议的 delipidation 和染色步骤的孵化时间。小组织的近似重量为 < 300 毫克。
辅助电影 1: 脂肪组织中交感神经支配的3D 影像学.酪氨酸羟化酶 (TH) 染色的 psWAT 样品 (如图 2所示)。这部电影显示了从 psWAT 的 dorsolumbar 和腹股沟地区的光学部分 (4X) 的飞行, 总深度为2毫米。自体荧光以洋红显示。该地区从 dorsolumbar 部分似乎有较低的 SNS 密度。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58271/JoVE_movie_1_R1.mp4">请单击此处下载此文件.</a>
补充电影 2: 3D 成像的血管在脂肪组织.CD31 染色的 psWAT 样本 (如图 3所示)。这部电影显示了从 psWAT 的 dorsolumbar 和腹股沟地区的光学部分 (4X) 的飞行, 总深度为2毫米. CD31 以红色显示。自体荧光显示在青色中。所有脂肪细胞似乎被毛细血管紧密包围。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58271/JoVE_movie_2_R1.mp4">请单击此处下载此文件.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biology

26.9K 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

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET成像的三维水凝胶

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

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

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

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 ...

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 ...

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

用于细胞表征和分泌特征的人类脂肪组织微分裂

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

单个细胞分辨率三维成像的有机体

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.