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A novel flat-embedding method to prepare ultrathin cryosections from cultured cells in their in situ orientation

By V. Oorschot, H. de Wit, W.G. Annaert and J. Klumperman


Immunogold labeling of ultrathin cryosections provides a sensitive and quantitative method to localize proteins at the ultrastructural level. An obligatory step in the routine preparation of cryosections from cultured cells is the detachment of cells from their substrate and subsequent pelleting. This procedure precludes visualization of cells in their\ud in situ orientation and hampers the study of polarized cells. Here we describe a method to\ud sample cultured cells from a petri dish or coverslip by embedding them in a 12% gelatin\ud slab. Subsequently, sections can be prepared in parallel or perpendicular to the plane of\ud growth. Our method extends the cryosectioning technique to applications in studying polarized cells and correlative light–electron microscopy

Topics: Geneeskunde, cryoultramicrotomy, immunogold, PC12 cells, hippocampal neurons, electron microscopy, flat-embedding
Year: 2002
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