A novel flat-embedding method to prepare ultrathin cryosections from cultured cells in their in situ orientation.
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.
Oorschot
Viola
V
Department of Cell Biology and Institute for Biomembranes, University Medical Center, Utrecht, The Netherlands.
de Wit
Heidi
H
Annaert
Wim G
WG
Klumperman
Judith
J
D000818:Animals; D016764:Cell Polarity; D002478:Cells, Cultured / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005629:Frozen Sections; D005780:Gelatin; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D016716:PC12 Cells; D050683:R-SNARE Proteins; D051381:Rats; D016610:Tissue Embedding
D008565:Membrane Proteins; D050683:R-SNARE Proteins; D005780:Gelatin