免疫标记肾组织的光学清除与成像
1Division of Nephrology and Clinical Immunology,University Hospital RWTH Aachen, 2III. Department of Medicine,University Medical Center, Hamburg-Eppendorf, 3Department of Nephrology,Monash Health, 4Division of Nephrology and Hypertension,Oregon Health and Science University, 5Renal Section,Veterans Affairs Portland Health Care System, 6Fondation LeDucq Transatlantic Networks of Excellence, 7Department of Internal Medicine, Nephrology and Transplantation,Erasmus Medical Center
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 在 1x 磷酸盐缓冲盐 (PBS) 中制作一批 3% 的甲醛 (PFA)。每只鼠标?…
Representative Results
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…
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0.22 µm filter | Fisher Scientific | 09-761-112 | |
| 15 mL conical tube | Fisher Scientific | 339650 | |
| 21 gauge butterfly needle | Braun | Venofix | |
| 3-way stopcock | Fisher Scientific | K420163-4503 | |
| 3D analyis software | Bitplane AG | IMARIS | |
| 3D analyis software | Cellprofiler | free open-source software | |
| 5-0 silk suture | Fine Science Tools | 18020-50 | |
| 50 ml plastic syringes | Fisher Scientific | 14-817-57 | |
| Anti-BrdU monoclonal antibody | Roche | 11296736001 | |
| Antibody diluent | Dako | S0809 | |
| CD31-647 | BioLegend | 102516 | |
| Citrate-based antigen retrieval solution | Vector Laboratories | H-3300 | |
| curved hemostat | Fisher Scientific | 13-812-14 | |
| Dako Wash Buffer | Agilent | S3006 | |
| dissecting microscope | Motic | DSK-500 | |
| Embedding cassettes | Carl Roth | E478.1 | |
| Ethanol | Merck | 100983 | |
| Ethyl cinnamate | Sigma-Aldrich | 112372 | |
| Flexible film/Parafilm M | Sigma-Aldrich | P7793 | |
| Goat anti-AQP2 | Santa Cruz Biotechnology | sc-9882 | |
| Guinea pig anti-NKCC2 | N/A | N/A | DOI: 10.1681/ASN.2012040404 |
| HCl | Carl Roth | P074.1 | |
| Heparin | Sagent Pharmaceuticals | 401-02 | |
| hemostat | Agnthos | 312-471-140 | |
| horizontal rocker | Labnet | S2035-E | |
| Imaging dish | Ibidi | 81218 | |
| Ketamine | MWI Animal Health | 501090 | |
| Micro serrefine | Fine Science Tools | 18052-03 | |
| NaOH | Fisher Scientific | S318-500 | |
| Operating scissors | Merit | 97-272 | |
| Paraformaldehyde | Thermo Fischer Scientific | O4042-500 | |
| Rabbit anti-phoshoThr53-NCC | PhosphoSolutions | p1311-53 | |
| Silicone elastomer | World Precision Instruments Kwik-Sil | KWIK-SIL | |
| Sodium azide | Sigma-Aldrich | S2002 | |
| Tissue slicer | Zivic Instruments | HSRA001-1 | |
| Triton X-100 | Acros Organics | AC215682500 | |
| Vannas scissors | Fine Science Tools | 15000-00 | |
| Vibratome | Lancer | Series 1000 | |
| Xylazine | MWI Animal Health | AnaSed Inj SA (Xylazine) |
References
- Oh, S. W., et al. A mesoscale connectome of the mouse brain. Nature. 508 (7495), 207-214 (2014).
- Zhai, X. Y., et al. 3-D reconstruction of the mouse nephron. Journal of the American Society of Nephrology. 17 (1), 77-88 (2006).
- Nyengaard, J. R. Stereologic methods and their application in kidney research. Journal of the American Society of Nephrology. 10 (5), 1100-1123 (1999).
- Puelles, V. G., Bertram, J. F., Moeller, M. J. Quantifying podocyte depletion: theoretical and practical considerations. Cell and Tissue Research. 369 (1), 229-236 (2017).
- Puelles, V. G., Moeller, M. J., Bertram, J. F. We can see clearly now: optical clearing and kidney morphometrics. Current Opinion in Nephrology and Hypertension. 26 (3), 179-186 (2017).
- Ariel, P. A beginner’s guide to tissue clearing. International Journal of Biochemistry and Cell Biology. 84, 35-39 (2017).
- Richardson, D. S., Lichtman, J. W. Clarifying Tissue Clearing. Cell. 162 (2), 246-257 (2015).
- Ke, M. T., Fujimoto, S., Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nature Neuroscience. 16 (8), 1154-1161 (2013).
- Kuwajima, T., et al. ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development. 140 (6), 1364-1368 (2013).
- Hama, H., et al. ScaleS: an optical clearing palette for biological imaging. Nature Neuroscience. 18 (10), 1518-1529 (2015).
- Susaki, E. A., et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell. 157 (3), 726-739 (2014).
- Chung, K., et al. Structural and molecular interrogation of intact biological systems. Nature. 497 (7449), 332-337 (2013).
- Lee, E., Sun, W. ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging. Journal of Visualized Experiments. (118), (2016).
- Klingberg, A., et al. Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy. Journal of the American Society of Nephrology. 28 (2), 452-459 (2017).
- Dodt, H. U., et al. Ultramicroscopy: 3-D visualization of neuronal networks in the whole mouse brain. Nature Methods. 4 (4), 331-336 (2007).
- Erturk, A., et al. 3-D imaging of solvent-cleared organs using 3-DISCO. Nature Protocols. 7 (11), 1983-1995 (2012).
- Becker, K., Jahrling, N., Saghafi, S., Weiler, R., Dodt, H. U. Chemical clearing and dehydration of GFP expressing mouse brains. PloS One. 7 (3), e33916 (2012).
- Renier, N., et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell. 159 (4), 896-910 (2014).
- Pan, C., et al. Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nature Methods. 13 (10), 859-867 (2016).
- Schwarz, M. K., et al. Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains. PLoS ONE. 10 (5), e0124650 (2015).
- Jing, D., et al. Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Research. 28 (8), 803-818 (2018).
- Staudt, T., Lang, M. C., Medda, R., Engelhardt, J., Hell, S. W. 2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microscopy Research and Technique. 70 (1), 1-9 (2007).
- Hama, H., et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nature Neuroscience. 14 (11), 1481-1488 (2011).
- Yang, B., et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell. 158 (4), 945-958 (2014).
- Hua, L., Zhou, R., Thirumalai, D., Berne, B. J. Urea denaturation by stronger dispersion interactions with proteins than water implies a 2-stage unfolding. Proceedings of the National Academy of Sciences of the United States of America. 105 (44), 16928-16933 (2008).
- Wan, P., et al. Evaluation of seven optical clearing methods in mouse brain. Neurophotonics. 5 (3), 035007 (2018).
- Puelles, V. G., et al. Validation of a 3-D Method for Counting and Sizing Podocytes in Whole Glomeruli. Journal of the American Society of Nephrology. 27 (10), 3093-3104 (2016).
- Puelles, V. G. Novel 3-D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis. Kidney International (in press). , (2019).
- Saritas, T., et al. Optical Clearing in the Kidney Reveals Potassium-Mediated Tubule Remodeling. Cell Reports. 25 (10), 2668-2675 (2018).
- Masselink, W., et al. Broad applicability of a streamlined ethyl cinnamate-based clearing procedure. Development. 146 (3), (2019).
- Clark, J. Z., et al. Representation and relative abundance of cell-type selective markers in whole-kidney RNA-Seq data. Kidney International. 95 (4), 787-796 (2019).
- Qi, Y., et al. FDISCO: Advanced solvent-based clearing method for imaging whole organs. Science Advances. 5 (1), eaau8355 (2019).
- Sylwestrak, E. L., Rajasethupathy, P., Wright, M. A., Jaffe, A., Deisseroth, K. Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution. Cell. 164 (4), 792-804 (2016).
- Shah, S., et al. Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing. Development. 143 (15), 2862-2867 (2016).
- Gleave, J. A., Lerch, J. P., Henkelman, R. M., Nieman, B. J. A method for 3-D immunostaining and optical imaging of the mouse brain demonstrated in neural progenitor cells. PLoS ONE. 8 (8), e72039 (2013).
- Saritas, T., et al. Disruption of CUL3-mediated ubiquitination causes proximal tubule injury and kidney fibrosis. Scientific Reports. 9 (1), 4596 (2019).
- Hall, A. M., Crawford, C., Unwin, R. J., Duchen, M. R., Peppiatt-Wildman, C. M. Multiphoton imaging of the functioning kidney. Journal of the American Society of Nephrology. 22 (7), 1297-1304 (2011).
- Holliger, P., Hudson, P. J. Engineered antibody fragments and the rise of single domains. Nature Biotechnology. 23 (9), 1126-1136 (2005).
- Bunka, D. H., Stockley, P. G. Aptamers come of age – at last. Nature Reviews: Microbiology. 4 (8), 588-596 (2006).
- Kim, S. Y., et al. Stochastic electrotransport selectively enhances the transport of highly electromobile molecules. Proceedings of the National Academy of Sciences of the United States of America. 112 (46), E6274-E6283 (2015).
- Liu, A. K. L., Lai, H. M., Chang, R. C., Gentleman, S. M. Free of acrylamide sodium dodecyl sulphate (SDS)-based tissue clearing (FASTClear): a novel protocol of tissue clearing for 3-D visualization of human brain tissues. Neuropathology and Applied Neurobiology. 43 (4), 346-351 (2017).
- Xu, N., et al. Fast free-of-acrylamide clearing tissue (FACT)-an optimized new protocol for rapid, high-resolution imaging of 3-D brain tissue. Scientific Reports. 7 (1), 9895 (2017).
- Tainaka, K., et al. Whole-body imaging with single-cell resolution by tissue decolorization. Cell. 159 (4), 911-924 (2014).
- Matryba, P., Bozycki, L., Pawlowska, M., Kaczmarek, L., Stefaniuk, M. Optimized perfusion-based CUBIC protocol for the efficient whole-body clearing and imaging of rat organs. Journal of Biophotonics. 11 (5), e201700248 (2018).
Citer Cet Article
Saritas, T., Puelles, V. G., Su, X., Ellison, D. H., Kramann, R. Optical Clearing and Imaging of Immunolabeled Kidney Tissue. J. Vis. Exp. (149), e60002, doi:10.3791/60002 (2019).