Name: adipo-clear: a tissue clearing method for three-dimensional imaging of adipose tissue
Uploaded: 2018-07-28T15:00:00
Description: Watch this Scientific Journal Video about Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue at JoVE.com

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?...

<ol>
	<li>Rosen, E. D., Spiegelman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+We+Talk+About+When+We+Talk+About+Fat.">What We Talk About When We Talk About Fat.</a> Cell. 156 (1-2), 20-44 (2014).</li><li>Barbatelli, G., 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=The+emergence+of+cold-induced+brown+adipocytes+in+mouse+white+fat+depots+is+determined+predominantly+by+white+to+brown+adipocyte+transdifferentiation.">The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.</a> American Journal of Physiology - Endocrinology and Metabolism. 298 (6), E1244-E1253 (2010).</li><li>Wang, Q. A., Tao, C., Gupta, R. K., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+adipogenesis+during+white+adipose+tissue+development,+expansion+and+regeneration.">Tracking adipogenesis during white adipose tissue development, expansion and regeneration.</a> Nature Medicine. 19 (10), 1338-1344 (2013).</li><li>Bartness, T. J., Liu, Y., Shrestha, Y. B., Ryu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+innervation+of+white+adipose+tissue+and+the+control+of+lipolysis.">Neural innervation of white adipose tissue and the control of lipolysis.</a> Frontiers in Neuroendocrinology. 35 (4), 473-493 (2014).</li><li>Morrison, S. F., Madden, C. J., Tupone, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+Neural+Regulation+of+Brown+Adipose+Tissue+Thermogenesis+and+Energy+Expenditure.">Central Neural Regulation of Brown Adipose Tissue Thermogenesis and Energy Expenditure.</a> Cell Metabolism. 19 (5), 741-756 (2014).</li><li>Xue, Y., 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=Hypoxia-Independent+Angiogenesis+in+Adipose+Tissues+during+Cold+Acclimation.">Hypoxia-Independent Angiogenesis in Adipose Tissues during Cold Acclimation.</a> Cell Metabolism. 9 (1), 99-109 (2009).</li><li>Shimizu, I., 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=Vascular+rarefaction+mediates+whitening+of+brown+fat+in+obesity.">Vascular rarefaction mediates whitening of brown fat in obesity.</a> The Journal of Clinical Investigation. 124 (5), 2099-2112 (2014).</li><li>Abe, H., 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=3D+reconstruction+of+brain+section+images+for+creating+axonal+projection+maps+in+marmosets.">3D reconstruction of brain section images for creating axonal projection maps in marmosets.</a> Journal of Neuroscience Methods. 286, 102-113 (2017).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Renier, N., Wu, Z., Simon, D. J., Yang, J., Ariel, P., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iDISCO:+A+Simple,+Rapid+Method+to+Immunolabel+Large+Tissue+Samples+for+Volume+Imaging.">iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging.</a> Cell. 159 (4), 896-910 (2014).</li><li>Renier, N., 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=Mapping+of+Brain+Activity+by+Automated+Volume+Analysis+of+Immediate+Early+Genes.">Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes.</a> Cell. 165 (7), 1789-1802 (2016).</li><li>Chi, J., 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=Three-Dimensional+Adipose+Tissue+Imaging+Reveals+Regional+Variation+in+Beige+Fat+Biogenesis+and+PRDM16-Dependent+Sympathetic+Neurite+Density.">Three-Dimensional Adipose Tissue Imaging Reveals Regional Variation in Beige Fat Biogenesis and PRDM16-Dependent Sympathetic Neurite Density.</a> Cell Metabolism. 27 (1), 226-236 (2018).</li><li>Khan, T., 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=Metabolic+Dysregulation+and+Adipose+Tissue+Fibrosis:+Role+of+Collagen+VI.">Metabolic Dysregulation and Adipose Tissue Fibrosis: Role of Collagen VI.</a> Molecular and Cellular Biology. 29 (6), 1575-1591 (2009).</li><li>Croce, A. C., Bottiroli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescence+Spectroscopy+and+Imaging:+A+Tool+for+Biomedical+Research+and+Diagnosis.">Autofluorescence Spectroscopy and Imaging: A Tool for Biomedical Research and Diagnosis.</a> European Journal of Histochemistry EJH. 58 (4), (2014).</li><li>Oh, D. Y., Morinaga, H., Talukdar, S., Bae, E. J., Olefsky, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Macrophage+Migration+Into+Adipose+Tissue+in+Obese+Mice.">Increased Macrophage Migration Into Adipose Tissue in Obese Mice.</a> Diabetes. 61 (2), 346-354 (2012).</li><li>Cinti, 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=Adipocyte+death+defines+macrophage+localization+and+function+in+adipose+tissue+of+obese+mice+and+humans.">Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.</a> Journal of Lipid Research. 46 (11), 2347-2355 (2005).</li><li>Wang, W., Seale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+brown+and+beige+fat+development.">Control of brown and beige fat development.</a> Nature Reviews Molecular Cell Biology. 17 (11), 691-702 (2016).</li></ol>

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&#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...

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 ...

Research

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

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, ...

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

一个系统为培养虹膜色素上皮细胞，研究蝾螈镜头再生

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

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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

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

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

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

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

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D 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 ...

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

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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 ...