Hawes P, Netherton C L, Mueller M, Wileman T, Monaghan P
Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.
J Microsc. 2007 May;226(Pt 2):182-9. doi: 10.1111/j.1365-2818.2007.01767.x.
We describe a method for high-pressure freezing and rapid freeze-substitution of cells in tissue culture which provides excellent preservation of membrane detail with negligible ice segregation artefacts. Cells grown on sapphire discs were placed 'face to face' without removal of tissue culture medium and frozen without the protection of aluminium planchettes. This reduction in thermal load of the sample/holder combination resulted in freezing of cells without visible ice-crystal artefact. Freeze-substitution at -90 degrees C for 60 min in acetone containing 2% uranyl acetate, followed by warming to -50 degrees C and embedding in Lowicryl HM20 gave consistent and clear membrane detail even when imaged without section contrasting. Preliminary data indicates that the high intrinsic contrast of samples prepared in this way will be valuable for tomographic studies. Immunolabelling sensitivity of sections of samples prepared by this rapid substitution technique was poor; however, reducing the uranyl acetate concentration in the substitution medium to 0.2% resulted in improved labelling. Samples substituted in this lower concentration of uranyl acetate also gave good membrane detail when imaged after section contrasting.
我们描述了一种用于组织培养中细胞高压冷冻和快速冷冻置换的方法，该方法能出色地保存膜细节，且冰分离伪像可忽略不计。生长在蓝宝石圆盘上的细胞在不除去组织培养基的情况下“面对面”放置，并且在没有铝制载片保护的情况下进行冷冻。样品/支架组合的热负荷降低，使得细胞冷冻时没有可见的冰晶伪像。在含有2%醋酸铀酰的丙酮中于-90℃进行60分钟的冷冻置换，然后升温至-50℃并包埋在Lowicryl HM20中，即使在没有切片对比成像的情况下也能得到一致且清晰的膜细节。初步数据表明，以这种方式制备的样品的高固有对比度对断层扫描研究将很有价值。通过这种快速置换技术制备的样品切片的免疫标记灵敏度较差；然而，将置换介质中醋酸铀酰的浓度降低到0.2%可提高标记效果。用这种较低浓度醋酸铀酰置换的样品在切片对比成像后也能呈现出良好的膜细节。
