Cryoimmobilization is an optimal method of preserving sample ultrastructure in electron microscopy studies. However, cryoimmobilization is limited to thin samples and this limitation may necessitate the isolation of the structure of interest. For cellular structures that are found in low number, or only during certain phases of the cell cycle, an added benefit of isolation is the possibility to concentrate the structures. We developed a method to perform correlative light and electron microscopy on infrequent isolated subcellular structures. In this chapter, we will describe our protocol that uses a combination of existing techniques and new solutions for the isolation, identification, cryoimmobilization, targeted ultramicrotomy, and imaging of the free-floating meiotic spindles assembled in Xenopus laevis egg extract.
Keywords: Electron tomography; High-pressure freezing; Isolated sample; Targeted ultramicrotomy.
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