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基于纳米黄金的蛋白质定位使SBF-SEM通过细胞结蛋白的亚细胞可视化

Feng-Xia Liang¹, Joseph Sall², Chris Petzold²

¹Microscopy Laboratory, Division of Advanced Research Technologies, New York University Grossman School of Medicine, New York, NY, United States; Department of Cell Biology, New York University Grossman School of Medicine, New York, NY, United States.

Methods in cell biology

|July 14, 2023

概括

我们开发了一个新的3D成像协议,使用预嵌入体积免疫电子显微镜 (预嵌入vIEM) 来精确地定位细胞中的蛋白质. 这种方法增强了超结构性保存,并减少了样本充电,以获得更清晰的纳米级成像.

科学领域:

细胞生物学细胞生物学
显微镜技术显微镜技术
生物物理学的生物物理.

背景情况:

光显微镜受到 difraktion 的限制,阻碍了详细的超结构分析.
体积电子显微镜 (vEM) 在3D中提供纳米分辨率,但需要优化分子定位协议.
精确的3D蛋白质定位对于理解细胞结构和功能至关重要.

研究的目的:

提出一种新的预嵌入体积免疫电子显微镜 (预嵌入vIEM) 协议.
为了在复杂组织中实现特定蛋白质的高分辨率3D超结构检测,例如Connexin 43.
克服传统免疫标签的局限性,包括标签透和组织保存.

主要方法:

开发了一种平衡样本固定,抗原/超结构保存和标签透的协议.
利用一个小的1.4纳米黄金标记器与银增强检测和减少样品充电.
为成功的串行块面扫描电子显微镜 (SBF-SEM) 样本准备所纳入的策略.

主要成果:

在3D中成功将Connexin 43定位在小鼠心脏组织的插曲盘中.
证明有效的抗原和超结构性保存,没有遗传编码的记者.
展示了SBF-SEM成像的减少样品充电和改进的切割.

结论:

基于小黄金的预嵌入vIEM协议非常适合在细胞区和膜微域内进行分子定位.
该方法为3D超结构分析和蛋白质映射提供了强大的方法.
该协议克服了先进电子显微镜免疫标记方面的关键挑战.

关键词:

连接器 43 连接器 43 子纳米金的黄金免疫电子显微镜的使用间隔性圆盘间隔性圆盘是指间隔性圆盘.纳米黄金是什么意思预嵌入体积免疫电子显微镜 (预嵌入vIEM)连续块面扫描电子显微镜.银的增强银的增强.卷电子显微镜的体积电子显微镜.

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