完整澄清器官中克 氏锥虫感染细胞、休眠无鞭毛虫和 T 细胞的定量 3D 成像
概要
本协议描述了光片荧光显微镜和自动化软件辅助方法，以可视化和精确量化完整，清除的器官和组织中的增殖和休眠 克氏锥虫 寄生虫和T细胞。这些技术提供了一种可靠的方法来评估治疗结果，并为寄生虫 – 宿主相互作用提供了新的见解。
Abstract
恰加斯病是一种被忽视的病理学，影响着全世界数百万人，主要是在拉丁美洲。恰加斯病病原体克 氏锥虫 （T. cruzi）是一种专性细胞内寄生虫，具有多种生物学特性，可感染包括人类在内的几种哺乳动物物种，引起心脏和消化系统病变。长期以来，人们需要对 克氏锥虫 体内感染进行可靠的检测，以了解恰加斯病的复杂生物学并准确评估治疗方案的结果。目前的协议展示了一种集成管道，用于在3D重建，清除的器官中自动定量克 氏锥虫感染的细胞。光片荧光显微镜可以准确地观察和量化整个器官或组织中活跃增殖和休眠的 克氏锥虫 寄生虫和免疫效应细胞。此外，还成功采用了CUBIC-HistoVision管道，用抗体和核染色剂对清除的器官进行统一标记。组织清除与3D免疫染色相结合，为全面评估药物治疗方案提供了一种公正的方法，提高了对 克氏锥虫感染组织的细胞组织的理解，并有望推进与抗克氏锥虫 免疫反应、组织损伤和恰加斯病修复相关的发现。
Introduction
由原生动物寄生虫克氏锥虫引起的恰加斯病是世界上最被忽视的热带疾病之一，每年造成约13，000人死亡。感染通常从急性期进展为慢性期，导致 30% 的患者出现心脏病，包括心律失常、心力衰竭和猝死1，2。尽管在急性期引起了针对寄生虫的强烈宿主免疫反应，但少量寄生虫在宿主的整个生命过程中长期存在于心脏和骨骼肌等组织中。有几个因素，包括适应性免疫反应的延迟发作和寄生虫的非复制形式的存在，可能有助于克氏锥虫避免被免疫系统完全消除的能力3，4，5，6。此外，寄生虫的非复制休眠形式显示出对锥虫杀虫药物的低敏感性，并且可能部分导致在许多情况下观察到的治疗失败7，8。
新成像技术的发展为深入了解寄生虫在感染组织中的空间分布及其与参与其控制的免疫细胞的关系提供了机会。这些特征对于更好地了解免疫系统控制寄生虫的过程和跟踪慢性组织中存在的罕见休眠寄生虫至关重要。
光片荧光显微镜（LSFM）是最全面和无偏的方法之一，无需薄切片即可对大型组织或器官进行3D成像。光片显微镜利用薄片光仅激发焦平面中的荧光团，减少样品的光漂白和光毒性，并使用超快相机记录数千个组织层的图像。通过在组织脱脂和脱色后均匀化折射率（RI），从而减少光的散射并呈现高质量的图像，从而获得激光在组织中正确穿透所需的高水平的组织透明度9，10，11。
已经开发了组织清除方法，用于对全小鼠12，13，14，类器官15，16，17，表达报告荧光标记物的器官18，19，20，21，22，23以及最近有限数量的人体组织24进行成像.目前的组织清除方法分为三类：（1）基于有机溶剂的方法，如DISCO协议25，26，（2）基于水凝胶的方法，如CLARITY 27，以及水性方法，如CUBIC（透明，无阻碍的脑/身体成像鸡尾酒和计算分析）18，19，28，29.CUBIC 方案保持器官形状和组织完整性，保留内源性表达报告蛋白的荧光。该技术的最新更新，CUBIC-HistoVision（CUBIC-HV），还允许使用荧光标记抗体和DNA标记检测表位28。
在本协议中，使用CUBIC管道检测在澄清的完整小鼠组织中表达荧光蛋白的克 氏锥虫 。对光学透明组织进行LSFM成像，3D重建，并自动定量每个器官的克 氏锥虫 感染细胞，休眠无鞭毛虫和T细胞的精确总数。此外，该协议已成功采用，以获得具有抗体和核染色的清除器官的统一标记。这些方法对于了解 克氏锥虫 在感染宿主中的扩增和控制至关重要，并且有助于全面评估恰加斯病的化学和免疫疗法。
Protocol
本研究严格按照《公共卫生服务实验动物人道关怀及使用政策》及实验动物护理评审协会评审及认证指引进行。动物使用协议（控制小鼠克 氏锥虫 感染-A2021 04-011-Y1-A0）已获得佐治亚大学机构动物护理和使用委员会的批准。B6.C+A2：A44g-Gt（ROSA）26Sor tm14（CAG-tdTomato）Hze/J，B6.Cg-Gt（ROSA）26Sortm14（CAG-tdTomato）Hze/J和C57BL/6J-Tg（Cd8a*-cre）B8Asin/J小鼠（雌性，1-2个月大）用于本研究。…
Representative Results
用PBS洗涤CUBIC固定组织以除去固定剂，然后与CUBIC-L鸡尾酒一起孵育，CUBIC-L鸡尾酒是一种氨基醇的基本缓冲溶液，可从组织中提取色素和脂质，导致组织脱色，同时保持组织结构。通过组织可以看到纸张中的网格线，表明器官的适当清除（图2A）。脱脂后，将组织洗涤并分别浸入CUBIC-R +和安装溶液中进行RI均质化和成像（图2B）。</strong…
Discussion
缺乏寄生虫的广泛全器官成像和免疫应答限制了对宿主-寄生虫相互作用复杂性的理解，并阻碍了对恰加斯病治疗的评估。本研究采用CUBIC管道对 克氏锥虫感染小鼠的完整器官和组织进行澄清和染色。
本研究测试了多种组织清除方案（PACT32、ECi 33、FLASH34、iDISCO11、26 和 fDISCO13</…
開示
The authors have nothing to disclose.
Acknowledgements
我们感谢Etsuo Susaki博士在组织清除和免疫染色方案方面的宝贵帮助和建议。此外，我们感谢CTEGD生物医学显微镜核心的M. Kandasamy使用LSFM和共聚焦成像的技术支持。我们还要感谢塔尔顿研究小组的所有成员在整个研究中提供的有用建议。
Materials
| 1-methylimidazole | Millipore Sigma | 616-47-7 | |
| 2,3-Dimethyl-1-phenyl-5-pyrazolone (Antipyrine | TCI | D1876 | |
| 6-wells cell culture plates | ThermoFisher Scientific | 140675 | |
| AlexaFluor 647 anti-mouse Fab fragment | Jackson Immuno Research Laboratories | 315-607-003 | |
| AlexaFluor 647 anti-rabbit Fab fragment | Jackson Immuno Research Laboratories | 111-607-003 | |
| anti-GFP nanobody Alexa Fluor 647 | Chromotek | gb2AF647-50 | |
| anti-RFP | Rockland | 600-401-379 | |
| anti-α-SMA | Sigma | A5228 | |
| B6.C+A2:A44g-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mouse | The Jackson Laboratory | Strain #007914 | Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D |
| B6.Cg-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze/J mouse | The Jackson Laboratory | Strain #007914 | Common Name: Ai14 , Ai14D or Ai14(RCL-tdT)-D |
| BOBO-1 Iodide | ThermoFisher Scientific | B3582 | |
| Bovine serum albumin (BSA) | Sigma | #A7906 | |
| C57BL/6J-Tg(Cd8a*-cre)B8Asin/J mouse | The Jackson Laboratory | Strain #032080 | Common Name: Cd8a-Cre (E8III-Cre) |
| CAPSO | Sigma | #C2278 | |
| Cleaning wipes Kimwipes | Kimberly-Clark | T8788 | |
| Confocal Laser Scanning Microscope | Zeiss | LSM 790 | |
| CUBIC-HV 1 3D immunostaining kit | TCI | C3699 | |
| CUBIC-HV 1 3D nuclear staining kit | TCI | C3698 | |
| CUBIC-L | TCI | T3740 | |
| CUBIC-P | TCI | T3782 | |
| CUBIC-R+ | TCI | T3741 | |
| Cyanoacrylate-based gel superglue | Scotch | 571605 | |
| DiR (DiIC18(7); 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide) Company: Biotium | Biotium | #60017 | |
| Ethylene diamine tetra acetic acid (EDTA) | Millipore Sigma | 60-00-4 | |
| Falcon Centrifuge tubes 15 mL | Corning | CLS430791 | |
| Falcon Centrifuge tubes 50 mL | Corning | CLS430290 | |
| Formalin | Sigma-Aldrich | HT501128 | |
| Heparin | ThermoFisher Scientific | J16920.BBR | |
| Hyaluronidase | Sigma | #H3884 or #H4272 | |
| Imaris File Converter x64 | BitPlane | v9.2.0 | |
| Imaris software | BitPlane | v9.3 | |
| ImSpector software | LaVision BioTec, Miltenyi Biotec | v6.7 | |
| Intravenous injection needle 23-G | Sartori, Minisart Syringe filter | 16534 | |
| Kimwipes | lint free wipes | ||
| Light-sheet fluorescent microscope | Miltenyi Biotec | ULtramicroscope II imaging system | |
| Methanol | ThermoFisher Scientific | 041838.K2 | |
| Micropipette tips, 10 µL, 200 µL and 1,000 µL | Axygen | T-300, T-200-Y and T-1000-B | |
| Motorized pipet dispenser | Fisher Scientific, Fisherbrand | 03-692-172 | |
| Mounting Solution | TCI | M3294 | |
| N-butyldiethanolamine | TCI | B0725 | |
| Nicotinamide | TCI | N0078 | |
| N-Methylnicotinamide | TCI | M0374 | |
| Paraformaldehyde (PFA) | Sigma-Aldrich | 158127 | |
| Phosphate buffered saline (PBS) | Thermo Fisher Scientific | 14190-094 | |
| RedDot 2 Far-Red Nuclear Stain | Biotium | #40061 | |
| Sacrifice Perfusion System | Leica | 10030-380 | |
| Scissors | Fine Science Tools | 91460-11 | |
| Serological pipettes | Costar Sterile | 4488 | |
| Shaking incubator | TAITEC | BR-43FM MR | |
| Sodium azide (NaN3) | ThermoFisher Scientific | 447815000 | |
| Sodium carbonate (Na2CO3) | ThermoFisher Scientific | L13098.36 | |
| Sodium Chloride (NaCl) | ThermoFisher Scientific | 447302500 | |
| Sodium hydrogen carbonate (NaHCO3) | ThermoFisher Scientific | 014707.A9 | |
| SYTOX-G Green Nucleic Acid Stain | ThermoFisher Scientific | S7020 | |
| Triton X-100 | Sigma-Aldrich | T8787 |
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