A Novel Approach for Intracellular 3D Immuno-Labeling for Electron Tomography

作者：Jimenez Nuria; Post Jan Andries^*

来源：Traffic, 2012, 13(7): 926-933.

DOI：10.1111/j.1600-0854.2012.01363.x

• cell monolayer
• chemical fixation
• electron microscopy
• electron tomography
• freeze substitution
• high-pressure freezing
• pre-embedment immuno-labeling
• rehydration
• targeted laser cell perforation

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.

• 出版日期2012-7