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Zusammenfassung:
The chapter discusses commonly used procedures for visualizing both fixed and live echinoderm embryos: (1) formaldehyde fixation of cleavage stage sea urchin embryos, (2) staining and imaging fixed embryos, (3) simultaneous fixation and visualization of the actin and microtubule cytoskeletons, (4) observation of live embryos, (5) 4-D imaging of fluorescent markers in live starfish oocytes, (6) protocol for imaging the dynamics of nuclear lamina during nuclear envelope breakdown, and (7) 4-D imaging of dextran entry during nuclear envelope breakdown (NEBD). Several procedures for fixing early sea urchin embryos with formaldehyde, cold methanol, detergents, and buffers are described. For staining and imaging echinoderm embryo, immunofluorescence is a widely used technique, as there are many commercially available antibodies that will cross-react beautifully with endogenous echinoderm proteins. General protocols for the immunofluorescence of formaldehyde-fixed cells and for staining nucleic acids with various small-molecule dyes are provided in the chapter. The embryos of echinoderms are amenable to live observation, accompanied by time-lapse video microscopy. The different aspects of the cells are emphasized using various imaging techniques, such as Differential Interference Contrast (DIC). Live embryos are easily observed as wet mounts on a standard slide or in a perfusion chamber, but the coverslip prevents direct access to the cells. A variety of chamber slides are described for live observation of echinoderm embryos.
