Name: three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary
Uploaded: 2017-12-10T14:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary at JoVE.com

这项研究得到了中国博士后 (No. 2014T70392 至 YF)、中国国家自然科学基金 (No. 81673766 至 YF)、新教师启动基金、复旦大学 Zuoxue 基金会的赠款和发展上海市高峰学科综合医学项目 (20150407)。

所有涉及动物的程序均遵循复旦大学上海医学院动物伦理委员会的指导方针 (批准编号 20160225-013)。
1. 透明小鼠卵巢的制备
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解决方案的准备
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<li>准备磷酸盐缓冲盐水 (PBS) 溶液 (1 米, pH 7.6) 与0.1% 海卫 X-100 (PBST)。制作 1 L 10x PBS 库存溶液, 混合87克氯化钠, 3.1 克 NaH2PO4 ·2H2O, 28.7 克 Na2HPO4 · 12H2o 添加 dH2</s...

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在目前的研究中, 我们提出3D 成像来评估毛细血管与个体生长卵泡之间的关系。在我们以前使用相同的协议9的工作中, 我们研究了大血管、卵泡之间的相互作用以及完整的小鼠卵巢中卵泡的位置。被动清晰的方法使我们能够研究微 vasculatures、卵泡、黄和卵泡之间的相互关系以及在不同发育阶段重建卵巢结构。
为了获得完整的组织的3D 图像数据, 清除过程是第...

卵泡是卵巢的基本结构和功能单位, 其发育与卵巢内的血管高度相关。血管为卵泡提供营养和荷尔蒙, 从而在卵泡的生长和成熟中发挥重要作用1。
包括选择性血管标记、转基因小鼠模型和药物开发在内的各种技术的结合, 增加了我们对卵巢血管网络、血管生成和血管功能的认识。卵泡.卵巢被称为活动器官, 因为它在卵泡和排卵期间改造各种组织和血管网络。在血管的大小和结构的这种积极重塑是需要的生物功能的发展和招募卵泡。
传统的组织学和计量方法使用的卵巢切片和 immunolabeling 的血管是有限的二维 (2D) 图像2。随着三维 (3D) 重建技术的发展, 2D 图像的组织切片可以重叠, 使一个3D 的结构, 但这种方法仍然有一些局限性-切片的组织可以破坏的显微构造, 部分组织往往是缺掉的, 重要的劳动参与了从切片获得的图像进行3D 重建。全组织3D 成像与共聚焦显微镜可以克服许多这些限制, 但这些方法仅限于对胚胎卵巢血管生成的评价3。使用完整的组织清除方法, 如清晰的4可以增加可视化体积, 以解决产后和成人卵巢中的这些问题, 这些方法提供无任何结构变形的卵巢的光清除。对完整子房的3D 结构进行成像, 为图像分析软件提供了一个精确的图像数据库, 如本工作中使用的 Imaris 软件包。
整个成年期的卵巢重塑是一个动态生理系统的一部分, 这使得卵巢成为一个很好的模型来研究血管生成的调节。此外, 评估卵巢血管在女性生殖系统的病理条件, 如多囊卵巢综合征或卵巢癌的作用, 可以通过整个卵巢组织成像进行研究。被动清晰度法的发展和先进的图像分析软件的使用, 提供了详细的空间信息的关系之间的血管和卵巢结构, 如卵泡。

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		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Acrylamide</td>
			<td style="width:189px;">Vetec</td>
			<td style="width:127px;">v900845</td>
			<td style="width:861px;">http://www.sigmaaldrich.com/catalog/product/vetec/v900845</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Flour 488 (Dilution 1:50)&#160;</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:127px;">A11039</td>
			<td style="width:861px;">https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Flour 594 (Dilution 1:50)</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:127px;">A11012</td>
			<td style="width:861px;">https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Bisacrylamide</td>
			<td style="width:189px;">Amresco</td>
			<td style="width:127px;">172</td>
			<td style="width:861px;">http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Black wall glass bottom dish (Willco-Dish)</td>
			<td style="width:189px;">Ted Pella</td>
			<td style="width:127px;">14032</td>
			<td style="width:861px;">http://www.tedpella.com/section_html/706dish.htm#black_wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Boric acid</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10004818</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10004818</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:487px;">Disodium hydrogen phosphate dodecahydrate (Na2HPO4 12H2O)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10020318</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10020318</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FocusClear</td>
			<td style="width:189px;">Celexplorer</td>
			<td style="width:127px;">FC-102</td>
			<td style="width:861px;">http://www.celexplorer.com/product_list.asp?MainType=107&#38;BRDarea=1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Parafilm</td>
			<td style="width:189px;">Bemis</td>
			<td style="width:127px;">PM996</td>
			<td style="width:861px;">http://www.parafilm.com/products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Paraformaldehyde</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">80096618</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=80096618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10)</td>
			<td style="width:189px;">Abcam</td>
			<td style="width:127px;">ab28364</td>
			<td style="width:861px;">http://www.abcam.com/cd31-antibody-ab28364.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Photoinitiator VA044</td>
			<td style="width:189px;">Wako</td>
			<td style="width:127px;">va-044/225-02111</td>
			<td style="width:861px;">http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium azide</td>
			<td style="width:189px;">Sigma</td>
			<td style="width:127px;">S2002</td>
			<td style="width:861px;">http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium chloride (NaCl)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10019318</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10019318</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:487px;">Sodium dihydrogen phosphate dihydrate (NaH2PO4 2H2O)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">20040718</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=20040718</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium dodecyl sulfate</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">30166428</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=30166428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium hydroxide (NaOH)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10019718</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10019718</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Triton X-100</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">30188928</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=30188928</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Tyrosine hydroxylase (TH, Dilution 1:50)</td>
			<td style="width:189px;">Abcam</td>
			<td style="width:127px;">ab76442</td>
			<td style="width:861px;">http://www.abcam.com/tyrosine-hydroxylase-phospho-s40-antibody-ab51206.html</td>
		</tr>
	</tbody>
</table>

three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary

卵巢是雌性生殖系统的主要器官, 对雌性配子的生产和内分泌系统的控制至关重要, 但复杂的结构关系和三维 (3D) 的血管结构卵巢不是很好描述。为了形象化3D 连接和结构的血管在完整的卵巢, 第一个重要的步骤是使卵巢光学清晰。为了避免组织萎缩, 我们使用了水凝胶固定的被动清晰度 (透明脂质交换丙烯酰胺-杂交刚性成像/免疫/原位杂交-相容组织水凝胶) 的协议方法清除完整的卵巢.免疫, 先进的多共聚焦显微镜, 和3D 图像重建, 然后用于可视化卵巢血管和卵泡毛细血管。采用这种方法, 我们发现卵泡毛细血管长度与卵泡壁容积之间存在显著的正相关 (P < 0.01)。

我们将无源清晰方法应用于无源卵巢清除的快速简便方法, 同时保留了卵泡和血管结构, 并从标记的血管和卵泡中获得最高的荧光信号。卵泡血管的3D 结构是由免疫为 CD31, 一个标记为内皮细胞6。用灯丝算法追踪成年小鼠卵巢中的 CD31 染色, 并显示卵泡毛细血管与初级和次级卵泡之间的相互关系 (图 1)。
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Watch this Scientific Journal Video about Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary at JoVE.com

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

在这里, 我们提出了被动清晰度和3D 重建方法的适应性, 可视化卵巢血管和卵泡毛细血管在完整的小鼠卵巢。

无源清清除小鼠卵巢血管构筑的三维重建

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Research

JoVE Journal

Biology

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

科研

生物学

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, ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

使用高解析度电脑断层可视化的三维结构和功能的植物血管

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

幻灯片制备方法，以保存睾丸细菌细胞的三维染色质结构

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

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 ...

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 ...