title: A novel approach for intracellular 3D immuno-labeling for electron tomography.
abstract: Electron tomography (ET) is an indispensable high-resolution tool for three dimensional (3D) imaging in cell biology. When applied to immuno-labeled cells, ET can provide essential insights in both the cellular architecture and the dynamics. Current protocols for 3D immuno-labeling of intracellular antigens include permeabilization steps that cause random, extensive cell membrane disruption. This permeabilization results in a poor cell ultrastructure, limiting the usefulness of the specimens for high-resolution studies. Here we describe a novel method, based on a well-controlled permeabilization by targeted laser cell perforation, that allows for the 3D immuno-localization of cytoplasmic antigens in cultured cells. The approach is unique since it is applicable to both chemically and cryo-fixed cells and leads to a superior ultrastructural preservation for electron microscopy and tomography.
authors.affiliation: Department of Biomolecular Imaging, Institute of Biomembranes, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
authors.forename: Nuria
authors.initials: N
authors.lastname: Jiménez
authors.forename: Jan Andries
authors.initials: JA
authors.lastname: Post
mesh_terms: D021941:Caveolae / Q000648:ultrastructure; D055032:Electron Microscope Tomography / Q000379:methods*; D061307:Human Umbilical Vein Endothelial Cells / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry; D007834:Lasers; D061251:Primary Cell Culture