Preparation of shadowed nuclear envelopes from Xenopus

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Abstract
Methods for examining the structure of the nuclear envelope of oocytes of Xenopus laevis by electron microscopy using metal shadowing have been developed and evaluated. Minor modifications were made to existing methods for preparing specimens by freeze drying, mainly to eliminate unnecessary steps and a rapid method for examining the structure and arrangement of nuclear envelope components, based on dehydration in an ethanol series followed by amyl acetate and then air drying, was also developed. The preservation of the lamina and connections between the nuclear pore complexes using the rapid air drying method was satisfactory for observing the fibrous components of the envelope and their attachment to the pores. Furthermore, air drying required only simple laboratory apparatus and, moreover, offered several advantages compared to freeze drying when assessing the effect of various disruptive treatments on the nuclear envelope or examining the connections between its components. In specimens prepared by either the more rapid air drying method or by freeze drying, the lamina meshwork beneath the nuclear face of the envelope was clear, but the fine structure of the nuclear pore complexes was superior in freeze dried preparations. In views of the nucleoplasmic face of the envelope, the lamina meshwork was suspended above the support film in freeze dried preparations, but collapsed in most air dried specimens. This collapse was not without its advantages, however, as it facilitated observation of the connections between nuclear pore complexes and lamina fibers, which were often masked in freeze dried preparations.