title: Correlative light and electron microscopy for a free-floating spindle in Xenopus laevis egg extracts.
abstract: 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.
authors.affiliation: Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, Oeiras, Portugal; European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Tranfield
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany; Institut Curie, Centre de Recherche, Paris, France; Structure and Membrane Compartments, CNRS UMR144, Paris, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Heiligenstein
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Peristere
authors.affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany; Institut de Génétique et Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, BP 10142, Illkirch, France.
authors.forename: Claude
authors.initials: C
authors.lastname: Antony
mesh_terms: D000818:Animals; D002457:Cell Extracts; D055032:Electron Microscope Tomography / Q000379:methods; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence / Q000379:methods; D008867:Microtomy; D009865:Oocytes / Q000648:ultrastructure; D008941:Spindle Apparatus / Q000648:ultrastructure*; D014982:Xenopus laevis
chemical_list: D002457:Cell Extracts
keywords: Electron tomography; High-pressure freezing; Isolated sample; Targeted ultramicrotomy