Name: whole-kidney three-dimensional staining with cubic
Uploaded: 2022-07-18T13:00:00
Description: Watch this Scientific Journal Video about Whole-Kidney Three-Dimensional Staining with CUBIC at JoVE.com

这项工作的一部分是通过与Hiroki R. Ueda教授（东京大学），Etsuo A. Susaki教授（顺天堂大学），Tetsuhiro Tanaka教授（东北大学），Masafumi Fukagawa教授，Takehiko Wada博士和Hirotaka Komaba博士（东海大学）合作进行的。

所有实验均获得东京大学机构审查委员会的批准。所有动物程序均根据美国国立卫生研究院指南进行。8周龄的雄性C57BL / 6NJcl小鼠用于本研究。小鼠是从商业来源获得的（见 材料表）。
1. 动物制备和肾脏固定
<ol><li>按照以下步骤进行灌注固定。<ol><li>通过吸入异氟醚（3%，2.0L / min）和腹膜内给予盐酸美托咪定（0.3mg / kg），酒石酸布托芬诺（5mg / kg）...

<ol>
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A., Ueda, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+and+whole-organ+clearing+and+imaging+techniques+with+single-cell+resolution:+toward+organism-level+systems+biology+in+mammals.">Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals.</a> Cell Chemical Biology. 23 (1), 137-157 (2016).</li><li>Keller, P. J., Dodt, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+microscopy+of+living+or+cleared+specimens.">Light sheet microscopy of living or cleared specimens.</a> Current Opinion in Neurobiology. 22 (1), 138-143 (2012).</li><li>Susaki, E. A., 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=Advanced+CUBIC+protocols+for+whole-brain+and+whole-body+clearing+and+imaging.">Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.</a> Nature Protocols. 10 (11), 1709-1727 (2015).</li><li>Kubota, S. 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=Whole-body+profiling+of+cancer+metastasis+with+single-cell+resolution.">Whole-body profiling of cancer metastasis with single-cell resolution.</a> Cell Reports. 20 (1), 236-250 (2017).</li><li>Hasegawa, 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=Comprehensive+three-dimensional+analysis+(CUBIC-kidney)+visualizes+abnormal+renal+sympathetic+nerves+after+ischemia/reperfusion+injury.">Comprehensive three-dimensional analysis (CUBIC-kidney) visualizes abnormal renal sympathetic nerves after ischemia/reperfusion injury.</a> Kidney International. 96 (1), 129-138 (2019).</li><li>Hasegawa, S., Inoue, T., Inagi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroimmune+interactions+and+kidney+disease.">Neuroimmune interactions and kidney disease.</a> Kidney Research and Clinical Practice. 38 (3), 282-294 (2019).</li><li>Hasegawa, 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=The+oral+hypoxia-inducible+factor+prolyl+hydroxylase+inhibitor+enarodustat+counteracts+alterations+in+renal+energy+metabolism+in+the+early+stages+of+diabetic+kidney+disease.">The oral hypoxia-inducible factor prolyl hydroxylase inhibitor enarodustat counteracts alterations in renal energy metabolism in the early stages of diabetic kidney disease.</a> Kidney International. 97 (5), 934-950 (2020).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Hasegawa, 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=Activation+of+sympathetic+signaling+in+macrophages+blocks+systemic+inflammation+and+protects+against+renal+ischemia-reperfusion+injury.">Activation of sympathetic signaling in macrophages blocks systemic inflammation and protects against renal ischemia-reperfusion injury.</a> Journal of the American Society of Nephrology. 32 (7), 1599-1615 (2021).</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=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>Klingberg, A., 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=Fully+automated+evaluation+of+total+glomerular+number+and+capillary+tuft+size+in+nephritic+kidneys+using+lightsheet+microscopy.">Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy.</a> Journal of the American Society of Nephrology. 28 (2), 452-459 (2017).</li><li>Zhao, 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=Cellular+and+molecular+probing+of+intact+human+organs.">Cellular and molecular probing of intact human organs.</a> Cell. 180 (4), 796-812 (2020).</li><li>Susaki, E. A., 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=Versatile+whole-organ/body+staining+and+imaging+based+on+electrolyte-gel+properties+of+biological+tissues.">Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.</a> Nature Communications. 11 (1), 1-22 (2020).</li></ol>

该协议允许对各种结构进行全肾3D成像，例如交感神经，集合管，动脉，近端小管和肾小球9，10，11。这种染色方法提供了宏观观察，并通过可视化缺血再灌注损伤9，10后肾交感神经的改变和糖尿病肾病初始阶段肾小球肿大11，带来了新的生物学发现。
<p class="jove...

肾脏由各种细胞群组成。虽然常规病理学为我们提供了许多关于肾脏微环境的信息，但需要三维（3D）成像来精确了解肾脏疾病进展过程中的细胞间串扰。过去，全器官3D成像需要进行大量的连续切片和图像重建1。然而，这种方法需要太多的努力，并且在重现性方面存在问题。
光学清除是克服这一障碍的好策略2，3。组织混浊主要是由于光散射和吸收，因为每个器官由各种物质组成，包括水、蛋白质和脂质。因此，组织清除的基本策略是用折射率（RI）匹配试剂代替组织中的水和脂质，这些试剂具有与蛋白质相同的光学特性4。为了观察透明标本，光片荧光显微镜是有用的5。光片从侧面照亮透明标本，激发信号通过位于垂直位置的物镜6采集。该显微镜在单次扫描中获取横截面信息，这与共聚焦或多光子荧光显微镜不同。因此，它可以快速获取具有低光漂白水平的z-stack图像。
清晰、无阻碍的脑/身体成像混合物和计算分析 （CUBIC） 是一种组织清除方法，可通过光片荧光显微镜2，7，8 进行全器官成像。在本研究中优化了立方体和全安装免疫荧光染色，以可视化小鼠肾脏3D结构9，10，11。使用这种全安装染色方法，在糖尿病肾病早期的缺血再灌注损伤 9，10 和肾小球肿大11 以及整个肾脏中的血管、近端小管和集合管9 后，可以看到肾交感神经的改变。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>14 mL Round Bottom High Clarity PP Test Tube</td>
			<td>Falcon</td>
			<td>352059</td>
			<td>Tissue clearing, staining, wash</td>
		</tr>
		<tr>
			<td>2,3-dimethyl-1-phenyl-5-pyrazolone/antipyrine</td>
			<td>Tokyo Chemical Industry</td>
			<td>D1876</td>
			<td>CUBIC-R+</td>
		</tr>
		<tr>
			<td>37%-Formaldehyde Solution</td>
			<td>Nacalai Tesque</td>
			<td>16223-55</td>
			<td>Post fixation</td>
		</tr>
		<tr>
			<td>4%-Paraformaldehyde Phosphate Buffer Solution</td>
			<td>Nacalai Tesque</td>
			<td>09154-85</td>
			<td>Kidney fixation</td>
		</tr>
		<tr>
			<td>Alexa Flour 555-conjugated donkey anti-sheep IgG antibody</td>
			<td>Invitrogen</td>
			<td>A-21436</td>
			<td>Secondary antibody (1:100)</td>
		</tr>
		<tr>
			<td>Alexa Flour 647-conjugated donkey anti-rabbit IgG antibody</td>
			<td>Invitrogen</td>
			<td>A-31573</td>
			<td>Secondary antibody (1:200)</td>
		</tr>
		<tr>
			<td>Anti-aquaporin 2 (AQP2) antibody</td>
			<td>Abcam</td>
			<td>ab199975</td>
			<td>Primary antibody (1:100)</td>
		</tr>
		<tr>
			<td>Anti-podocin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>P0372</td>
			<td>Primary antibody (1:100)</td>
		</tr>
		<tr>
			<td>Anti-sodium glucose cotransporter 2 (SGLT2) antibody</td>
			<td>Abcam</td>
			<td>ab85626</td>
			<td>Primary antibody (1:100)</td>
		</tr>
		<tr>
			<td>Anti-tyrosine hydroxylase (TH) antibody</td>
			<td>Abcam</td>
			<td>ab113</td>
			<td>Primary antibody (1:100)</td>
		</tr>
		<tr>
			<td>Anti-&#945;-smooth muscle actin (&#945;-SMA) antibody</td>
			<td>Abcam</td>
			<td>ab5694</td>
			<td>Primary antibody (1:200)</td>
		</tr>
		<tr>
			<td>Blocker Casein in PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>37528</td>
			<td>Staining buffer</td>
		</tr>
		<tr>
			<td>Butorphanol Tartrate</td>
			<td>Meiji</td>
			<td>005526</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>C57BL/6NJcl</td>
			<td>Nippon Bio-Supp.Center</td>
			<td>N/A</td>
			<td>Mouse strain</td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td>N/A</td>
			<td>Imaging analysis software</td>
		</tr>
		<tr>
			<td>Macro-zoom microscope</td>
			<td>OLYMPUS</td>
			<td>MVX10</td>
			<td>The observation unit of the custom-built microscope</td>
		</tr>
		<tr>
			<td>Medetomidine Hydrochloride</td>
			<td>Kyoritsu-Seiyaku</td>
			<td>008656</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>SANDOZ</td>
			<td>27803229</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td>Immersion oil</td>
		</tr>
		<tr>
			<td>N-buthyldiethanolamine</td>
			<td>Tokyo Chemical Industry</td>
			<td>B0725</td>
			<td>CUBIC-L, CUBIC-R+</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Tokyo Chemical Industry</td>
			<td>N0078</td>
			<td>CUBIC-R+</td>
		</tr>
		<tr>
			<td>Polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100</td>
			<td>Nacalai Tesque</td>
			<td>12967-45</td>
			<td>CUBIC-L, PBST</td>
		</tr>
		<tr>
			<td>Silicon oil HIVAC-F4</td>
			<td>Shin-Etsu Chemical</td>
			<td>50449832</td>
			<td>Immersion oil</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Wako</td>
			<td>195-11092</td>
			<td>Staining buffer</td>
		</tr>
	</tbody>
</table>

whole-kidney three-dimensional staining with cubic

尽管常规病理学提供了大量关于肾脏微观结构的信息，但由于缺乏三维（3D）信息，很难知道肾脏中血管，近端小管，集合管，肾小球和交感神经的精确结构。光学清除是克服这一巨大障碍的好策略。通过结合组织清除和3D成像技术，可以以单细胞分辨率分析整个器官中的多个细胞。然而，用于全器官成像的细胞标记方法仍然不发达。特别是，由于抗体穿透困难，整个安装器官染色具有挑战性。本协议开发了一种全安装小鼠肾脏染色，用于使用CUBIC（透明，无阻碍的脑/身体成像混合物和计算分析）组织清除方法进行3D成像。该方案能够从综合的角度可视化糖尿病肾病早期缺血再灌注损伤和肾小球肿大后的肾交感神经去神经。因此，这种技术可以通过提供宏观视角来导致肾脏研究的新发现。

使用这种染色方法，可视化整个肾脏中的交感神经[抗酪氨酸羟化酶（TH）抗体]和动脉[抗α平滑肌肌动蛋白（αSMA）抗体]（图4A，B和视频1）9。缺血/再灌注损伤（IRI）后也可见异常的肾交感神经9，10（图4C）。此外，使用该协议实现了整个脾脏13中?...

Watch this Scientific Journal Video about Whole-Kidney Three-Dimensional Staining with CUBIC at JoVE.com

Whole-Kidney Three-Dimensional Staining with CUBIC

本协议描述了用于三维（3D）肾脏成像的组织清除方法和全安装免疫荧光染色。这种技术可以为肾脏病理学提供宏观视角，从而带来新的生物学发现。

用立方体进行全肾三维染色

Although conventional pathology provided numerous information about kidney microstructure, it was difficult to know the precise structure of blood ...

该协议允许可视化整个肾脏中的三维结构，这可以通过提供宏观视角导致肾脏研究的创新发现。由于抗体穿透困难，整个器官染色具有挑战性。该方案已经证明了抗体对肾脏的高质量、全安装染色。首先，在灌注固定和肾脏采样后立即进行浸入固定，将肾脏浸入4摄氏度的4%多聚甲醛中16小时。使用 PBS 洗涤三次，每次两小时。通过将固定肾脏浸入7毫米的50%CUBIC-L中，在室温下轻轻摇动来进行脱色和脱脂过程。六小时后，将肾脏浸入 14 毫米圆底管中的 7 毫升 CUBIC-L 中，在 37 摄氏度下轻轻摇晃五天。每天刷新立方体-L。脱色和脱脂完成后，使用分配勺子处理样品，并在室温下用PBS洗涤肾脏三次，持续两小时。免疫染色脱脂肾在染色缓冲溶液中稀释的一抗，在37摄氏度的培养箱摇床中轻轻摇动7天。然后，在室温下用PBS（也称为PBST）中的0.5%Triton X-100清洗肾脏一天。对于二抗染色，在染色缓冲液中用二抗在黑暗中治疗肾脏，在37摄氏度下轻轻摇动7天。在室温下用PBST洗涤液固定染色一天。固定后，将肾脏浸入PB中的1%甲醛中3小时，并在室温下用PBS洗涤6小时。通过将肾脏浸入7毫升50%CUBIC-R +中，在14毫升圆底管中振荡一天，然后用7毫升CUBIC-R +在室温下摇动两天，进行折射率匹配。在免疫染色方法的帮助下，在3D肾脏成像中可视化整个肾脏中的交感神经和动脉。此外，缺血再灌注损伤后可见异常的肾交感神经。该方案还有助于可视化整个脾脏中的交感神经。此处显示了三维免疫荧光染色的数据定量过程。此外，在整个肾脏中成功可视化集合导管、近端小管的 S1 段和肾小球。在糖尿病肾病早期阶段给药引起的抑制性肾小球肿大也在3D成像中可视化。该协议可以标记整个肾脏中的特定分子。因此，研究人员可以分析脉管系统的结构改变或发现肾组织中的罕见事件。

Research

JoVE Journal

Medicine

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

三维成像伤害表皮内神经纤维在人体皮肤活检

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

科研

医学

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Three-Dimensional 3D Tumor Spheroid Invasion Assay

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

人垂体腺瘤细胞的三维海藻酸钠珠文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Three-Dimensional Printing of a Complex Aortic Anomaly

复杂主动脉异常的三维打印

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Estimation of Nephron Number in Whole Kidney using the Acid Maceration Method

酸化浸渍法评价全肾肾素数

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

三维打印指南模板辅助皮椎间盘（PVP）

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

三维胶原基质支架植入作为肝脏再生策略

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...

Guided Endodontics: Three-Dimensional Planning and Template-Aided Preparation of Endodontic Access Cavities

引导牙髓病学：牙髓通路腔的三维规划和模板辅助制备

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, ...