High-Resolution Scanning Electron Microscopy and Immuno-Gold Labeling of the Nuclear Lamina and Nuclear Pore Complex
Scanning electron microscopy (SEM) is a technique used to image surfaces. Field emission SEMs (feSEMs) can resolve structures that are ~0.5–1.5 nm apart. FeSEM, therefore is a useful technique for imaging molecular structures that exist at surfaces such as membranes. The nuclear envelope consists of four membrane surfaces, all of which may be accessible for imaging. Imaging of the cytoplasmic face of the outer membrane gives information about ribosomes and cytoskeletal attachments, as well as details of the cytoplasmic peripheral components of the nuclear pore complex, and is the most easily accessed surface. The nucleoplasmic face of the inner membrane is easily accessible in some cells, such as amphibian oocytes, giving valuable details about the organization of the nuclear lamina and how it interacts with the nuclear pore complexes. The luminal faces of both membranes are difficult to access, but may be exposed by various fracturing techniques. Protocols are presented here for the preparation, labeling, and feSEM imaging of Xenopus laevis oocyte nuclear envelopes.
Key wordsNuclear envelope Nuclear pore complex Scanning electron microscopy Nuclear lamina
Thanks to Christine Richardson, Helen Grindley, Pam Ritchie, and Sandra Rutherford for technical assistance, Terry Allen, Herbert Macgregor, Reimer Stick, and the late Rob Apkarian for help in developing these methods, and the Biotechnology and Biological Sciences Research Council, UK, grant number BB/G011818/1 for funding.