电子显微镜下细胞内放射自显影的方法。

M H Silk, A O Hawtrey, I M Spence, J H Gear
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引用次数: 27

摘要

介绍了一种在电子显微镜下进行高分辨率细胞内放射自显影的技术。将LLC-MK(2)猴肾细胞在含有0.4微居里/ ml胸苷- h(3)的培养基中培养72小时。标记后,用四氧化锇固定细胞,并包埋在甲基丙烯酸酯中。标记组织的超薄切片在formvar涂层和碳稳定的电子显微镜网格上拍摄。然后将一层银金属蒸发到组织上。涂覆的网格在红光下暴露在溴蒸气中1.5至2分钟,允许干燥1分钟,然后用1%的含水明胶薄膜覆盖,通过在玻璃钉上支撑的网格上降低的细线环施加。为了进行放射自显影,网格在4摄氏度的避光容器中与氯化钙干燥剂一起储存50天。在20℃下用1:1稀释的Promicrol (May and Baker, England)进行5分钟显影,然后用水冲洗1分钟,在15%的硫代硫酸钠水溶液中固定2.5分钟。在37℃的水中浸泡16小时去除明胶后,将自射线图干燥并在电子显微镜下检查。超微结构细节相当清晰,每个标记细胞的细胞质上覆盖着一层电子不透明的银沉积物,这表明含有胸腺嘧啶的多核苷酸可能在细胞质中合成。这件事被讨论了。
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A METHOD FOR INTRACELLULAR AUTORADIOGRAPHY IN THE ELECTRON MICROSCOPE.

A technic is described for high resolution intracellular autoradiography in the electron microscope. Cultures of LLC-MK(2) monkey kidney cells were incubated for 72 hours in a medium containing 0.4 microcurie per ml of thymidine-H(3). After labeling, the cells were fixed with osmium tetroxide and embedded in methacrylate. Ultrathin sections of the labeled tissue were taken up on Formvar-coated and carbon-stabilized electron microscope grids. A 150 to 450 A layer of silver metal was then evaporated onto the tissue. The coated grids were exposed to bromine vapor for 1.5 to 2 minutes under red light, allowed to dry for 1 minute, and then covered with a thin film of 1 per cent aqueous gelatin applied by means of a fine wire loop lowered over the grid supported on a glass peg. For autoradiographic exposure, the grids were stored 50 days in a light-proof container at 4 degrees C with calcium chloride desiccant. Development was carried out for 5 minutes at 20 degrees C in Promicrol (May and Baker, England) diluted 1:1 with water, followed by a 1 minute water wash and fixation for 2.5 minutes in 15 per cent aqueous sodium thiosulphate. After removal of the gelatin by immersion for 16 hours in water at 37 degrees C, the autoradiograms were dried and examined in the electron microscope. Ultrastructural detail was fairly well defined and the cytoplasm of each labeled cell was covered with an electron opaque deposit of silver, suggesting that a polynucleotide containing thymidine may be synthesized in the cytoplasm. The matter is discussed.

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