Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique

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Author: Robenek, H
Author: Severs, NJ
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Methods Article

Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique

Horst Robenek¹^* and Nicholas J. Severs²

¹ Department of Cell Biology and Ultrastructure Research, Leibniz-Institute for Arteriosclerosis Research. University of Münster, Domagkstr. 3D-48149 Münster. Germany.
² National Heart and Lung Institute, Imperial College London. U.K..

* To whom correspondence should be addressed: Horst Robenek, Department of Cell Biology and Ultrastructure Research, Leibniz-Institute for Arteriosclerosis Research. University of Münster, Domagkstr. 3D-48149 Münster. Germany. Phone: +49-251-83-56426. Fax: +49-251-83-52998. Email: robenek@uni-muenster.de

Biol. Proced. Online 2008;10:9-19. doi:10.1251/bpo138
Submitted: October 18, 2007; Accepted: December 12, 2007; Published: January 28, 2008.

Indexing terms: Freeze Fracturing; Microscopy, Immunoelectron; Immunogold Techniques.

Figure 1 Enlarged

Fig. 1:

The key steps in FRIL. Tissue or cell samples are rapidly frozen and fractured (1 and 2). The freeze-fracture process splits the lipid bilayer (2) exposing the fracture face. A platinum-carbon replica is made of the fractured specimen (3). The replica is treated with SDS to remove the cellular components apart from those attached to the replica (4). Proteins still attached to the replica are then immunogold labeled. On examination in the electron microscope, the electron-dense gold label is clearly visible against the replica, marking the target molecule in the membrane plane.

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