Name: optical clearing and imaging of immunolabeled kidney tissue
Uploaded: 2019-07-22T14:00:00
Description: Watch this Scientific Journal Video about Optical Clearing and Imaging of Immunolabeled Kidney Tissue at JoVE.com

T. S. 得到 DFG 德国研究基金会 (332853055)、埃尔塞·克鲁纳-弗雷塞纽斯-斯蒂夫通 (2015_A197) 和 RWTH Aachen 医学院(RWTH 返回计划)的资助。V.G.P.由德国格塞尔舍夫毛皮尼腓罗基、亚历山大·冯·洪堡基金会和澳大利亚国家健康和医学研究委员会的研究奖学金提供支持。D. H. E 由 LeDucq 基金会支持。R. K. 得到 DFG(KR-4073/3-1、SCHN1188/5-1、SFB/TRR57、SFB/TRR219)、北莱茵威斯特法伦州(MIWF-NRW)和RWTH Aachen大学跨学科临床研究中心(O3-11)的资助。

注:此处描述的所有实验程序均获得美国俄勒冈州波特兰市俄勒冈健康与科学大学机构动物护理和使用委员会 (IACUC) 以及德国 Aachen 相关地方当局的批准。
1. 逆行腹部主动脉灌注和小鼠肾脏固定
<ol><li>准备解决方案的前一天或晚上,并储存在冰箱过夜。使用前加热至室温 (RT) 的解决方案。</li>
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在 1x 磷酸盐缓冲盐 (PBS) 中制作一批 3% 的甲醛 (PFA)。每只鼠标?...

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光学清除技术在各种器官的三维可视化和微解剖学定量方面得到了广泛的关注。在这里,溶剂型清除方法(ECi)与免疫标签相结合,用于肾切片中整个小管的三维成像。此方法简单、便宜且快速。然而,其他研究问题最好用其他结算协议5来回答。同样重要的是要记住,溶剂型方法导致组织收缩在可变程度,主要是由于脱水步骤14,18。大多数溶剂?...

研究整个器官或大型多细胞结构的兴趣日益浓厚,因此发展了光学清除方法,涉及三维透明组织的成像。直到最近,估计整个结构的细胞数、长度或体积的最佳方法是立体学或详尽的序列切片,它基于组织的系统采样,以便随后在两个维度1中进行分析。2,3.然而,这些方法非常耗时,需要高水平的培训和专业知识4。光学清除方法克服了这些问题,通过平衡整个样品的折射率,使组织半透明为3-D成像5,6,7。
已开发几种光学清除方法,主要分为两类:溶剂型和水基法。水基方法可进一步分为简单浸没8、9、超水化10、11和水凝胶嵌入12、13。溶剂型方法使组织脱水,去除脂质,并将折射率标准化至1.55左右。大多数溶剂型方法的局限性是淬火常用报告蛋白的内源性荧光,如GFP、溶剂毒性、某些成像室或物镜中使用的溶解胶的能力,以及组织在脱水14,15,16,17,18,19,20,21。然而,溶剂型方法简单、高效,可在许多不同的组织类型中工作。
基于水性的方法依赖于组织浸入水溶液中,其折射率在1.38-1.528、11、12、22、23、24之间.这些方法是为了保持内源荧光报告器蛋白的排放和防止脱水引起的收缩而开发的,但大多数基于水性清除方法的局限性包括延长协议持续时间、组织扩张和蛋白质修饰(即在高保湿协议中,如ScaleA2)7、11、23、25中,尿素对蛋白质进行部分变性。ScaleS通过结合尿素与山梨醇来解决组织扩张问题,通过脱水尿素引起的组织扩张来平衡,并保存了电子显微镜10评估的组织超微结构。组织收缩或扩张影响结构的绝对大小、物体之间的距离或每个体积的细胞密度;因此,在组织清理时测量大小变化可能有助于解释获得的结果7,26。
通常,光学清除协议由多个步骤组成,包括预处理、渗透、免疫标记(如果需要)、折射率匹配以及使用高级光显微镜成像(例如,双光子、共聚焦或光片荧光显微镜)。大多数的清除方法已经开发可视化神经元组织,和新兴的研究已经验证了其在其他器官的应用5。这个综合工具已经证明,允许可靠和高效的肾脏结构分析,包括肾功能质27,28,免疫渗透28,血管28,和管状段29,它是一种理想的方法,以更好地了解肾小球功能和管状重塑在健康和疾病。
这里总结的是一种溶剂型方法,结合了肾小管的免疫染色;光学清除与廉价,无毒,和现成的化学乙基锡化酯(ECi);和共聚焦显微镜成像,允许完整的小管可视化和定量。这种方法很简单,将肾切片的抗原回收与商业抗体的染色相结合,不需要专门的设备,这使得大多数实验室都能使用。

<table>
	<tbody>
		<tr>
			<td>0.22 &#181;m filter</td>
			<td>Fisher Scientific</td>
			<td>09-761-112</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>Fisher Scientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>21 gauge butterfly needle</td>
			<td>Braun</td>
			<td>Venofix</td>
			<td></td>
		</tr>
		<tr>
			<td>3-way stopcock</td>
			<td>Fisher Scientific</td>
			<td>K420163-4503</td>
			<td></td>
		</tr>
		<tr>
			<td>3D analyis software</td>
			<td>Bitplane AG</td>
			<td>IMARIS</td>
			<td></td>
		</tr>
		<tr>
			<td>3D analyis software</td>
			<td>Cellprofiler</td>
			<td></td>
			<td>free open-source software</td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>Fine Science Tools</td>
			<td>18020-50</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml plastic syringes</td>
			<td>Fisher Scientific</td>
			<td>14-817-57</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-BrdU monoclonal antibody</td>
			<td>Roche</td>
			<td>11296736001</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody diluent</td>
			<td>Dako</td>
			<td>S0809</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31-647</td>
			<td>BioLegend</td>
			<td>102516</td>
			<td></td>
		</tr>
		<tr>
			<td>Citrate-based antigen retrieval solution</td>
			<td>Vector Laboratories</td>
			<td>H-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>curved hemostat</td>
			<td>Fisher Scientific</td>
			<td>13-812-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Wash Buffer</td>
			<td>Agilent</td>
			<td>S3006</td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Motic</td>
			<td>DSK-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding cassettes</td>
			<td>Carl Roth</td>
			<td>E478.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl cinnamate</td>
			<td>Sigma-Aldrich</td>
			<td>112372</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible film/Parafilm M</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-AQP2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-9882</td>
			<td></td>
		</tr>
		<tr>
			<td>Guinea pig anti-NKCC2</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>DOI: 10.1681/ASN.2012040404</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Carl Roth</td>
			<td>P074.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent Pharmaceuticals</td>
			<td>401-02</td>
			<td></td>
		</tr>
		<tr>
			<td>hemostat</td>
			<td>Agnthos</td>
			<td>312-471-140</td>
			<td></td>
		</tr>
		<tr>
			<td>horizontal rocker</td>
			<td>Labnet</td>
			<td>S2035-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging dish</td>
			<td>Ibidi</td>
			<td>81218</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>MWI Animal Health</td>
			<td>501090</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro serrefine</td>
			<td>Fine Science Tools</td>
			<td>18052-03</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating scissors</td>
			<td>Merit</td>
			<td>97-272</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Thermo Fischer Scientific</td>
			<td>O4042-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-phoshoThr53-NCC</td>
			<td>PhosphoSolutions</td>
			<td>p1311-53</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>World Precision Instruments Kwik-Sil</td>
			<td>KWIK-SIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue slicer</td>
			<td>Zivic Instruments</td>
			<td>HSRA001-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Acros Organics</td>
			<td>AC215682500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Lancer</td>
			<td>Series 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>MWI Animal Health</td>
			<td>AnaSed Inj SA (Xylazine)</td>
			<td></td>
		</tr>
	</tbody>
</table>

optical clearing and imaging of immunolabeled kidney tissue

光学清除技术通过在样本中平衡折射率,从而使组织透明,以便进行后续的三维(3-D)成像。他们在所有研究领域都备受关注,因为有可能分析跨越宏观距离的微观多细胞结构。鉴于肾小管、血管、神经和肾小球向多个方向延伸,而迄今为止,传统二维技术仅部分捕获了这些小球,组织清除也开辟了肾脏研究的许多新领域。光学清除方法列表正在迅速增加,但该领域的初学者仍然难以为给定的研究问题选择最佳方法。这里提供了一个简单的方法,结合抗体标记厚小鼠肾片;光学清除,具有廉价、无毒和即用化学乙基辛酸酯;和共聚焦成像。该协议描述了如何注入肾脏并使用抗原检索步骤来增加抗体结合,而无需专门的设备。其应用在肾脏内不同的多细胞结构成像中,并解决了组织中抗体渗透不良的问题。我们还讨论了成像内源性荧光道和获取非常大的样本的潜在困难以及如何克服它们。这个简单的协议提供了一个易于设置和全面的工具,以研究三维组织。

肾脏是复杂的器官,由43种不同的细胞类型组成31。这些细胞大多形成大型多细胞结构,如球红蛋白和管状物,其功能高度依赖于相互作用。经典的二维组织学技术部分地捕捉了这些大型结构,并可能错过完整结构中的焦移31。因此,使用光学清除技术进行三维分析有助于了解它们在健康和疾病中的作用。
大多数溶剂型光学清除技术至少会部分淬火...

Watch this Scientific Journal Video about Optical Clearing and Imaging of Immunolabeled Kidney Tissue at JoVE.com

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

免疫标记肾组织的光学清除与成像

抗体标记、光学清除和高级光显微镜相结合,可对完整的结构或器官进行三维分析。本文介绍的是将厚肾切片的免疫标记、光学清除与乙酰辛纳酯和共聚焦成像相结合的简单方法,可实现三维肾脏结构的可视化和定量。

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

对研究大型多细胞结构的兴趣日益增长，导致这种光学清除技术的发展，使透明器官内的大型结构能够进行三维可视化和定量。该协议提供了一个简单，便宜，易于设置和全面的工具，以调查组织在三个维度。传统的二维技术不能总是模拟疾病的多层性质。组织三维分析有助于更好地诊断和监测疾病。此方法不限于肾脏组织，可应用于任何正常和患病组织。对于任何在动物处理方面有一定经验的科学家来说，这个简单的协议都很容易理解。在抗体标签不良的情况下测试不同的抗体非常重要，并且只进行二级抗体控制。首先根据手稿说明在 1X PBS 中制备含有 PBS 和 3.0% 甲醛的肝素，并将溶液转移到单独的 50 mL 塑料注射器中。甲醛是有毒的，应在烟气罩中准备。根据手稿指示收集和大量肾脏。然后，取出胶囊，将肾脏切成一毫米厚的日冕片。将肾片与准备好的甲醛溶液一起浸入15 mL锥形管中。固定后，用1X洗涤缓冲液在水平摇杆上洗洗肾片两次，然后进行抗原检索。在 500 mL 烧杯中加热 300 mL 的 1X 抗原解质溶液。然后，将肾片封闭在可渗透到加热缓冲液的嵌入盒中，并在 92 至 98 摄氏度下搅拌一小时。对于此抗原检索步骤，保持温度稳定在 92 至 98 摄氏度之间非常重要。然后将切片转移到 10 mL 的 1X 洗涤缓冲液中，用 0.1% Triton X-100 进行摇动。第二天，用10 mL的新鲜1X洗涤缓冲液洗两次切片一小时。在500微升正常抗体稀释剂中稀释原抗体。在37摄氏度下，在稀释的原抗体中轻轻摇动肾片四天。孵育后，在室温下隔夜在10 mL的1X洗涤缓冲液中洗肾片，8小时后更换缓冲液一次。在500微升正常抗体稀释剂中稀释二级抗体，并在37摄氏度下用肾片孵育四天。请注意，从这一步前进，肾脏切片应受到保护，免受光线。使用二次抗体孵育后，在室温下隔夜用10 mL 1X洗涤缓冲液洗肾片。要脱水肾片，将其转移到5mL的高档100%乙醇，并在室温下摇动两小时，一小时后刷新乙醇。将肾片浸入两 mL 的乙基辛纳酸酯中，并轻轻摇动过夜。当准备好成像组织时，将600至1000微升乙基辛纳酸酯加入玻璃底盘，并将肾片转移到盘中。在肾片上放置一个圆形盖滑，用石蜡膜密封盘子。将盘子放在显微镜成像平台上，根据手稿指示对组织进行成像。该协议已用于成功执行肾脏组织的三：D分析，并评估细胞功能和相互作用。该方法可能导致荧光记者淬火，但其他强荧光蛋白可能抵制信号淬火。腹主动脉扩散可改善抗体扩散。在某些情况下，抗体渗透不良会导致表面信号。这可以通过更高的浓度或补充抗体来纠正。如果感兴趣的抗原在肾脏血管中表达，应考虑血管内分娩。然而，针对输卵管脱皮细胞的蛋白的抗体不会穿过球状过滤屏障，并导致非特异性信号。组织可以标记与抗体检测特定的细胞群体或可视化整个管段。此外，通过组合多种抗体，可以实现3D蛋白质的合并。重要的是要记住，抗体标签可以通过良好的肾脏灌注，使用抗原检索步骤，和高抗体浓度得到改善。一些强报告蛋白可能会抵制此协议后荧光信号的淬火。要对此进行测试，请跳过抗原检索和抗体标记步骤。该技术尊重结构的三维性质，开辟了许多新的研究领域。它消除了与传统二维分析相关的必需假设和干扰。

Research

JoVE Journal

Medicine

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

双模式成像，皮肤组织氧合和血管功能

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

科研

医学

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

婴幼儿的脑代谢和血流动力学的非侵入式光学测量

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

肩胛间褐色脂肪组织的切伦科夫发光成像

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

玫瑰红Photothrombosis共聚焦光学成像<em&gt;在体内</em&gt;：单支脑卒中模型

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Murine Kidney Transplant Technique

小鼠肾脏移植技术

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

3D 超声成像: 快速、经济高效的肌骨骼组织形态分析

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

18F-fdg Petct 对活性人棕色脂肪组织的全身和区域定量

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

板基检测血液中一氧化氮的 vasp 磷酸化

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

通过物理和各向异性扩张对人体组织部分的纳米成像

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, ...