Grief C, Nermut M V, Hockley D J
Electron Microscopy and Photography Section, National Institute for Biological Standards and Control, Potters Bar, Herts, U.K.
Micron. 1994;25(2):119-28. doi: 10.1016/0968-4328(94)90036-1.
Rapid freezing, freeze substitution and low temperature embedding were used to obtain resin-embedded specimens of HIV and SIV for morphological and immunolabelling studies, with particular emphasis on the 'lateral bodies' and p6 protein. HIV- or SIV-infected cells were fixed in 3% paraformaldehyde and cryoprotected with 0.5 M sucrose. Cells were applied to pieces of Whatman No 1 filter paper and impact-frozen onto a liquid nitrogen cooled copper block. Specimens were freeze-substituted at -90 degrees C using one of three different media: (a) absolute methanol, (b) methanol containing 0.5% uranyl acetate, and (c) methanol containing glutaraldehyde, osmium tetroxide and uranyl acetate. Specimens substituted in methanol and uranyl acetate showed both good structural preservation and retention of antigenicity. We found that the use of filter paper for supporting the specimen was an important factor in obtaining good freezing rates and was more practical than freezing mixtures of cells and gelatin. When compared with specimens prepared by conventional fixation and embedding, freeze-substituted virus particles showed a greater uniformity of shape and size and were more dense in appearance. Distinct 'lateral bodies' were not observed in freeze-substituted viruses. The viral protein p6 was widely distributed in the centre of mature virus particles.
采用快速冷冻、冷冻置换和低温包埋的方法，制备了用于形态学和免疫标记研究的HIV和SIV树脂包埋标本，特别着重于“侧体”和p6蛋白。将感染HIV或SIV的细胞用3%的多聚甲醛固定，并用0.5 M蔗糖进行冷冻保护。将细胞铺在Whatman No 1滤纸片上，冲击冷冻到液氮冷却的铜块上。标本在-90℃下使用三种不同的介质之一进行冷冻置换：（a）无水甲醇，（b）含0.5%醋酸铀的甲醇，（c）含戊二醛、四氧化锇和醋酸铀的甲醇。用甲醇和醋酸铀置换的标本显示出良好的结构保存和抗原性保留。我们发现，使用滤纸片支撑标本是获得良好冷冻速率的一个重要因素，并且比冷冻细胞和明胶的混合物更实用。与通过传统固定和包埋制备的标本相比，冷冻置换后的病毒颗粒形状和大小的均匀性更高，外观上更致密。在冷冻置换的病毒中未观察到明显的“侧体”。病毒蛋白p6广泛分布于成熟病毒颗粒的中心。
