Abstract：
Continuing progress in super-resolution microscopy enables the study of sub-chromosomal chromatin organization in single cells with unprecedented detail. Here we describe refined methods for pulse-chase replication labeling of individual chromosome territories (CTs) and replication domain units in mammalian cell nuclei, with specific focus on their application to three-dimensional structured illumination microscopy (3D-SIM). We provide detailed protocols for highly efficient electroporation-based delivery or scratch loading of cell-impermeable fluorescent nucleotides for live-cell studies. Furthermore, we describe the application of (2\'S)-2\'-deoxy-2\'-fluoro-5-ethynyluridine (F-ara-EdU) and 5-vinyl-2\'-deoxyuridine (VdU) for the in situ detection of segregated chromosome territories and sister chromatids with minimized cytotoxic side effects.
摘要：
超分辨率显微镜的持续进展使得能够以前所未有的细节研究单细胞中的亚染色体染色质组织。在这里，我们描述了用于哺乳动物细胞核中单个染色体区域（CT）和复制域单元的脉冲追踪复制标记的精细方法。特别关注它们在三维结构照明显微镜（3D-SIM）中的应用。我们提供了用于活细胞研究的高效基于电穿孔的递送或细胞不可渗透的荧光核苷酸的划痕加载的详细方案。此外，我们描述了（2\'S）-2\'-脱氧-2\'-氟-5-乙炔基尿苷（F-ara-EdU）和5-乙烯基-2\'-脱氧尿苷（VdU）在原位检测分离的染色体区域和姐妹染色单体中的应用，并具有最小化的细胞毒性副作用。
