Abstract：
This protocol describes the fluorescence in situ hybridization (FISH) of DNA probes on mitotic chromosome spreads optimized for two super-resolution microscopy approaches-structured illumination microscopy (SIM) and stimulated emission depletion (STED). It is based on traditional DNA FISH methods that can be combined with immunofluorescence labeling (Immuno-FISH). This technique previously allowed us to visualize ribosomal DNA linkages between human acrocentric chromosomes and provided information about the activity status of linked rDNA loci. Compared to the conventional wide-field and confocal microscopy, the quality of SIM and STED data depends a lot more on the optimal specimen preparation, choice of fluorophores, and quality of the fluorescent labeling. This protocol highlights details that make specimens suitable for super-resolution microscopy and tips for good imaging practices.
摘要：
该协议描述了针对两种超分辨率显微镜方法优化的有丝分裂染色体上的DNA探针的荧光原位杂交（FISH）-结构化照明显微镜（SIM）和受激发射消耗（STED）。它基于传统的DNAFISH方法，可以与免疫荧光标记（Immuno-FISH）结合使用。该技术先前使我们能够可视化人类顶心染色体之间的核糖体DNA连接，并提供有关连锁rDNA基因座的活动状态的信息。与传统的宽视场和共聚焦显微镜相比，SIM和STED数据的质量更多地取决于最佳的标本制备，荧光团的选择，和荧光标记的质量。该协议突出细节，使标本适合超分辨率显微镜和提示良好的成像实践。
