Woods P S, Ledbetter M C, Tempel N
Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
J Electron Microsc Tech. 1991 Jun;18(2):183-91. doi: 10.1002/jemt.1060180214.
We describe methods for freezing and drying EDTA-expanded, fixed metaphase chromosomes and nuclei, attached to grids as whole-mounts, for transmission electron microscopy. These methods use a special apparatus that is simple to construct. While separate freezers and dryers are commercially available, one for freezing blocks of tissue by slamming them against a cold metal surface, and the other for vacuum drying the frozen tissue, our apparatus is designed for gentler, cryogenic liquid plunge freezing and drying, sequentially, in the same apparatus, thus avoiding any compression or damage to the specimen. Use of a cryoprotectant is not essential; however, good results are obtained more often when 20% ethanol is used. Freezing is accomplished by rapid propulsion of the grid, with specimens attached, into slushy N2 (-210 degrees C) within the drying chamber; drying is automatic, by either sublimation under vacuum or by solvent substitution using absolute ethanol followed by acetone, which, in turn, is removed with a critical-point dryer. The apparatus offers a means of drying chromosomes and nuclei in an expanded state, and avoids the shrinkage of these structures that occurs during stepwise passage through increasing concentrations of ethanol or acetone.
我们描述了用于冷冻干燥附着在网格上作为整装标本的经乙二胺四乙酸（EDTA）扩增的固定中期染色体和细胞核的方法，用于透射电子显微镜观察。这些方法使用一种易于构建的特殊装置。虽然市面上有单独的冷冻机和干燥机，一种是通过将组织块猛击在冷金属表面来冷冻组织块，另一种是用于真空干燥冷冻组织，但我们的装置设计用于在同一装置中依次进行更温和的低温液体骤冷冷冻和干燥，从而避免对标本造成任何压缩或损伤。使用冷冻保护剂并非必不可少；然而，当使用20%乙醇时，更常获得良好的结果。冷冻是通过将附着有标本的网格快速推进到干燥室内的淤浆状氮气（-210摄氏度）中来完成的；干燥是自动的，通过真空升华或使用无水乙醇然后丙酮进行溶剂置换，然后用临界点干燥器除去丙酮。该装置提供了一种干燥处于扩增状态的染色体和细胞核的方法，并避免了在逐步通过浓度不断增加的乙醇或丙酮过程中这些结构发生的收缩。
