High-resolution field emission scanning electron microscope imaging of internal cell structures after Epon extraction from sections: a new approach to correlative ultrastructural and immunocytochemical studies.
The availability of high-resolution field emission scanning electron microscopes (FESEM) and the recent development of a less destructive method for extracting Epon from sections motivated us to investigate these techniques for the study of internal cell structures. We chose the nuclear pore complex (NPC) and insect striated muscle as test objects. Chemically fixed or rapidly cryoimmobilized samples were embedded in Epon 812. The Epon was extracted from 200- to 300-nm-thick sections with a modified potassium methoxide-crown ether complex. The samples were viewed with high-resolution FESEM at low voltages. In tangential sections of isolated nuclear envelopes from Xenopus oocytes the cytoplasmic and intranuclear components ("fishtraps") of NPCs appeared identical to what has been described from whole mounts. In cross sections, fishtraps are seen in side view, which is possible only with this technique. In longitudinal and cross sections of insect flight muscle the classical arrangement of myofilaments and cross-bridges is well preserved. This method now makes it possible to image internal cell structures from any desired angle by high-resolution FESEM. Immunolabeling studies on the rabbit psoas muscle demonstrated that antigenicity of alpha-actinin was retained in Epon-extracted sections. Immunogold labeling with antibodies against alpha-actinin conjugated to 3-nm gold beads was intense, highly specific, and restricted to the Z lines. This method can overcome the penetration problem of immunogold labeling, since any cell component can be positioned at the surface of the section. Obviously this approach can become a powerful new tool for many areas of structural cell biology.
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Department of Zoology, University of Wisconsin, Madison 53706.
Malecki
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D000185:Actinin / Q000378:metabolism; D000818:Animals; D002477:Cells / Q000378:metabolism / Q000648:ultrastructure*; D003584:Cytological Techniques; D004330:Drosophila; D004853:Epoxy Resins; D005260:Female; D005426:Flight, Animal; D007150:Immunohistochemistry; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D009132:Muscles / Q000378:metabolism / Q000648:ultrastructure; D009685:Nuclear Envelope / Q000648:ultrastructure; D009865:Oocytes / Q000648:ultrastructure; D011817:Rabbits; D014981:Xenopus
D004853:Epoxy Resins; D000185:Actinin; C004875:EPON