使用前 Vivo 共聚焦显微镜可视化淋巴节点结构和细胞定位
Summary
该协议描述了一种在不改变器官结构的情况下在排空淋巴结中成像不同细胞群的技术。
Abstract
淋巴结(LUN)是体内传播的器官,与先天免疫反应可以连接适应性免疫。事实上,LN战略性地插在淋巴血管的路径中,允许组织抗原与LN中所有常驻免疫细胞亲密接触。因此,使用外体全LN成像了解细胞组成、分布、位置和相互作用,将增进有关人体如何协调局部和全身免疫反应的知识。该协议显示了在体内给荧光标记抗体进行体内给药后,使用传统的共聚焦显微镜和库存试剂,允许非常可重复且易于执行的方法的体外成像策略。通过皮下抗体注射,可以在排空LUN中标记不同的细胞群,而不影响可能由传统免疫荧光显微镜技术破坏的组织结构。
Introduction
淋巴结 (Lun) 是卵形器官,广泛存在于全身,具有桥接先天和自适应免疫反应的关键功能。LUN过滤淋巴,以识别外来颗粒和癌细胞,以对它们进行免疫反应1。抗原呈现细胞(ApCs),T细胞和B细胞一起产生抗原特异性抗体(体液免疫)和细胞毒性淋巴细胞(细胞免疫),以消除外来颗粒和癌细胞2。因此,了解淋巴系统中免疫细胞的动态对疫苗开发和癌症免疫治疗具有重要影响。
强大的显微镜的出现-包括新的共聚焦和超分辨率显微镜-使一个非凡的进步,在理解不同的免疫细胞群体如何在他们的原生环境中如何运行3。现在,可以使用与转基因小鼠结合探针,在特定靶点4、5的控制下表达荧光蛋白,可以成像几个同时发生的细胞亚型。事实上,高维技术,包括大规模细胞学和多参数流分析,对于扩大我们对健康和疾病中不同免疫细胞的分块和功能的知识至关重要。7.然而,为了准备这些技术的样本,组织需要消化,细胞从自然环境中分离出来,在细胞悬浮液中进行分析。为了超越这些限制,并允许生物学更好的翻译,这里提出的协议的目标是应用一个简单的方法,使用库存共聚焦显微镜图像外整个淋巴结,有利于提高速度,组织与传统的免疫荧光染色相比,结构保存和细胞存活。通过这种方法,我们能够证明缺乏β-T细胞的小鼠,一种参与宿主早期防御病原体4的T淋巴细胞的亚型,与野生型小鼠相比,已经破坏了毛囊和T细胞区。这些发现使我们能够进行一项研究,其中我们证明βT细胞在淋巴器官平衡和体液免疫反应4中起关键作用。此外,该协议为探针和抗体到达淋巴结提供了一个生理途径,因为它们在原位标记使用的报告的基础上,通过组织淋巴环流进行皮下施用并消散与抗体可视化淋巴相关结构8,9, 芽中心动力学10,11,12,和目标容易进入血流13 ,14,15.
Protocol
Representative Results
Discussion
成像与其他技术(包括分子生物学和高维免疫分型)的结合增强了我们在其原生环境中研究免疫细胞的能力。事实上,虽然其他方法可能需要组织消化和细胞分离(这可能导致组织完整性的丧失),但使用体内或前体成像在以地理方式调查不同细胞亚型方面具有很大优势3,16.转基因小鼠菌株的可得性迅速增加,这种菌株专门针对细胞表达不同的荧光。重要的是,报?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH(R01 AI43458至H.L.W.)的支持。
Materials
| BV421 anti-CD4 | BD Horizon | 562891 | GK1.5; 0.2 mg mL-1 |
| BB515 anti-CD19 | BD Horizon | 564509 | 1D3; 0.2 mg mL-1 |
| BB515 Rat IgG2a, κ Isotype Control | BD Horizon | 564418 | R35-95; 0.2 mg mL-1 |
| BV421 Mouse IgG2b, K Isotype Control | BD Horizon | 562603 | R35-38 0.2 mg mL-1 |
| Cellview culture dish | Greiner-Bio | 627861 | 35×10 mm with glass bottom |
| Insulin syringes | BD Plastipak | – | Insulin U-100 |
| Kimwipes | Kimtech Science Brand | 7557 | size 21 x 20 cm / 100 sheets per box |
| Microsurgery curved forceps | WEP Surgical Instruments | custom made | 12.5 cm |
| Microsurgery curved scissors | WEP Surgical Instruments | custom made | 11.5 cm |
| Needle | BD PrecisionGlide | – | 30 gauge × ½ inch |
| Nikon Eclipse Te + A1R confocal head | Nikon | – | loaded with main 4 laser lines (405, 488, 543 and 647 nm) |
| PE anti-F4/80 | BD Pharmigen | 565410 | T45-2342; 0.2 mg mL-1 |
| PE Rat IgG2a, κ Isotype Control | BD Pharmigen | 553930 | R35-95; 0.2 mg mL-1 |
| Zeiss LSM 710 confocal microscope | Zeiss | – | loaded with main 4 laser lines (405, 488, 543 and 647 nm) |
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