Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands.
Nat Protoc. 2011 Jul 28;6(8):1221-8. doi: 10.1038/nprot.2011.365.
In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in their physiological setting is thus an important aspect of adult stem cell research. Here we describe a protocol to produce sections (150-200 μm) of near-native tissue with optimal tissue and cellular morphology by avoiding artifacts inherent in standard freezing or embedding procedures. The activity of genetically expressed fluorescent proteins is maintained, thereby enabling high-resolution three-dimensional (3D) reconstructions of fluorescent structures in virtually all types of tissues. The procedure allows immunofluorescence labeling of proteins to depths up to 50 μm, as well as a chemical 'Click-iT' reaction to detect DNA-intercalating analogs such as ethynyl deoxyuridine (EdU). Generation of near-native sections ready for imaging analysis takes approximately 2-3 h. Postsectioning processes, such as antibody labeling or EdU detection, take up to 10 h.
近年来，许多小鼠模型被开发出来，使用荧光蛋白来标记和追踪成年细胞群体的命运。因此，在生理环境下对这些荧光标记物进行高分辨率可视化是成体干细胞研究的一个重要方面。在这里，我们描述了一种方案，通过避免标准冷冻或包埋过程中固有的伪影，来产生具有最佳组织和细胞形态的近天然组织切片（150-200μm）。遗传表达的荧光蛋白的活性得以维持，从而能够对几乎所有类型的组织中的荧光结构进行高分辨率三维（3D）重建。该方案允许对蛋白质进行免疫荧光标记，深度可达 50μm，还可以进行化学“Click-iT”反应，以检测如 5-乙炔基-2-脱氧尿苷（EdU）等嵌入 DNA 的类似物。准备用于成像分析的近天然切片的生成大约需要 2-3 小时。切片后处理，如抗体标记或 EdU 检测，最多需要 10 小时。
