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Journal of Histochemistry and Cytochemistry, Vol. 50, 1067-1080, August 2002, Copyright © 2002, The Histochemical Society, Inc.

ARTICLE

A Novel Flat-embedding Method to Prepare Ultrathin Cryosections from Cultured Cells in Their In Situ Orientation

Viola Oorschota, Heidi de Wita, Wim G. Annaertb, and Judith Klumpermana
a Department of Cell Biology and Institute for Biomembranes, University Medical Center, Utrecht, The Netherlands and Center for Biomedical Genetics, The Netherlands
b Laboratory for Neuronal Cell Biology, Center for Human Genetics, Flanders Interuniversity Institute for Biotechnology, Gasthuisberg-KU, Leuven, Belgium

Correspondence to: Judith Klumperman, Dept. of Cell Biology, AZU Rm G02.525, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: J.Klumperman@lab.azu.nl

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 in situ orientation and hampers the study of polarized cells. Here we describe a method to sample cultured cells from a petri dish or coverslip by embedding them in a 12% gelatin slab. Subsequently, sections can be prepared in parallel or perpendicular to the plane of growth. Our method extends the cryosectioning technique to applications in studying polarized cells and correlative light–electron microscopy.

(J Histochem Cytochem 50:1067–1080, 2002)

Key Words: cryoultramicrotomy, immunogold, PC12 cells, hippocampal neurons, electron microscopy, flat-embedding


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