高压冷冻-冷冻替代技术在神经组织超微结构中的应用
张 宾，吴亮亮，吴莹莹，潘立君，娄森森，孔 妤*
(中国科学院神经科学研究所，神经科学国家重点实验室，电镜技术平台，上海200031)
摘 要 采用高压冷冻-冷冻替代技术对小鼠和线虫这两种模式生物的神经组织进行了样品制备、超薄切片和电镜观察，并在取材方式上进行了摸索，旨在比较化学固定法（CF）、高压冷冻固定法（HPF）和化学-高压冷冻联合固定法（CF+HPF）三种不同方式对神经组织超微结构的影响，促进高压冷冻-冷冻替代技术在神经生物学中的应用。透射电镜观察结果显示：与传统的化学固定相比，高压冷冻固定对神经组织样品具有较好的优势，能够减少化学处理所产生的人工假象；另一方面，对于不同动物的神经组织而言，所适用的固定方式也不相同，鼠脑适用于活检枪（biopsy gun）取样后直接进行HPF固定，所得结构比较完整，神经组织微观结构清晰。而在高压冷冻之前选用CF进行预固定，能加大样品结构保存的完整性，但在膜的细腻程度上不如直接进行高压冷冻制样的结果。线虫样品由于存在较厚的体壁保护，它更适于直接进行HPF处理，从而缩短了固定前的滞留时间，可有效保留腹部神经元超微结构。
关键词 高压冷冻-冷冻替代；神经组织；活检枪；鼠脑；线虫
中图分类号：Ｑ３３６；Ｒ３３８ 文献标识码：A doi:10.3969/j.issn.1000-6281.2017.01.008
The application of highpressure freezing-freeze substitution technique in ultrastructure of nervous tissue
ZHANG Bin,WU Liang-liang, WU Ying-ying, PAN Li-jun, LOU Sen-sen, KONG Yu*
（Institute of Neuroscience, State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 200031,China）
Abstract To promote the application of high pressure freezing-freeze substitution (HPF-FS) technique in neurobiology, the ultrastructure of nervous tissue of two model organisms (mouse and nematode) were investigated via the sample preparation, ultrathin-sectioning and TEM observation. Besides, the influences of three different fixation methods including chemical fixation(CF), high pressure freezing (HPF) and the combinationfixation (CF+FS) to the ultrastructure of nervous tissue were further explored.The results of TEM demonstratethat HPF provides more advantages for nervous tissue and could greatly minimize the artificial illusion to the ultrastructure which given rise to traditional chemical fixatives. In addition, it was also found that the suitable fixation way varies from different nervous tissues. For example, it is suitable for mouse brain tissue that directly to befixed by HPF after biopsy sampling. The resulting ultrastructure is better than CF or CF+HPF in both completeness and exquisite degree of membrane, indicating that there is no need to use chemical fixatives before HPF fixation. Due to its thick integument, the nematode is more suitable to be fixed by direct HPF treatment so as to shorten the residence time before fixation and effectively maintain the ultrastructure of its abdominal neurons.
Keywords high pressure freezing-freezing substitution; nervous tissue; biopsy gun; mouse brain; nematode
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