Meehl Janet B, Giddings Thomas H, Winey Mark
Department of MCD Biology, University of Colorado, Boulder, CO, USA.
Methods Mol Biol. 2009;586:227-41. doi: 10.1007/978-1-60761-376-3_12.
Preservation of Tetrahymena thermophila basal body ultrastructure for visualization by transmission electron microscopy is improved by a combination of high pressure freezing (HPF) and freeze substitution (FS). These methods also reliably retain the antigenicity of cellular proteins for immuno-electron microscopy, which enables the precise localization of green fluorescent protein (GFP)-tagged and native basal body proteins. The plastic-embedded samples generated by these methods take full advantage of higher resolution visualization techniques such as electron tomography. We describe protocols for cryofixation, FS, immunolabeling, and staining. Suggestions for trouble shooting and evaluation of specimen quality are discussed. In combination with identification and manipulation of a rapidly expanding list of basal body-associated gene products, these methods are being used to increase our understanding of basal body composition, assembly, and function.
通过高压冷冻（HPF）和冷冻置换（FS）相结合的方法，可改善嗜热四膜虫基体超微结构的保存，以便通过透射电子显微镜进行观察。这些方法还能可靠地保留细胞蛋白的抗原性用于免疫电子显微镜，从而能够精确地定位绿色荧光蛋白（GFP）标记的和天然的基体蛋白。通过这些方法生成的塑料包埋样本充分利用了更高分辨率的可视化技术，如电子断层扫描。我们描述了冷冻固定、冷冻置换、免疫标记和染色的方案。讨论了故障排除和标本质量评估的建议。结合对快速增加的一系列基体相关基因产物的鉴定和操作，这些方法正被用于增进我们对基体组成、组装和功能的理解。
