Hagen C, Grünewald K
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany.
J Microsc. 2008 May;230(Pt 2):288-96. doi: 10.1111/j.1365-2818.2008.01987.x.
A method is described employing microcarrier spheres of cross-linked dextran for obtaining ultra- and semithin vitreous sections from high-pressure frozen anchorage-dependent (mammalian) cells. Avoiding trypsination or scraping cells off from the culture surface, the presented approach allows for cryoimmobilization, cryosectioning and cryoelectron microscopy/tomography of frozen-hydrated cells in an unperturbed manner which is important to preserve the native state of, for instance, the cytoskeleton. Furthermore, our studies on the 'life cycle' of Herpes simplex virus in Vero cells demonstrate that cell monolayers on microcarrier beads are well suited for fluorescence microscopic characterization of the sample prior to high-pressure freezing.
本文描述了一种方法，该方法使用交联葡聚糖微载体球，从高压冷冻的贴壁依赖性（哺乳动物）细胞中获取超薄和半薄玻璃切片。该方法避免了胰蛋白酶消化或从培养表面刮下细胞，能够以不受干扰的方式对冷冻水合细胞进行冷冻固定、冷冻切片和冷冻电子显微镜/断层扫描，这对于保存例如细胞骨架的天然状态非常重要。此外，我们对单纯疱疹病毒在Vero细胞中的“生命周期”的研究表明，微载体珠上的细胞单层非常适合在高压冷冻之前对样品进行荧光显微镜表征。
