XB-ART-41582
Cold Spring Harb Protoc
2010 May 01;20105:pdb.prot5427. doi: 10.1101/pdb.prot5427.
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High-magnification in vivo imaging of Xenopus embryos for cell and developmental biology.
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Embryos of the frog Xenopus laevis are an ideal model system for in vivo imaging of dynamic biological processes, from the inner workings of individual cells to the reshaping of tissues during embryogenesis. Their externally developing embryos are more amenable to in vivo analysis than internally developing mammalian embryos, and the large size of the embryos make them particularly suitable for time-lapse analysis of tissue-level morphogenetic events. In addition, individual cells in Xenopus embryos are larger than those in other vertebrate models, making them ideal for imaging cell behavior and subcellular processes (e.g., following the dynamics of fluorescent fusion proteins in living or fixed cells and tissues). Xenopus embryos are amenable to simple manipulations of gene function, including knockdown and misexpression, and the large number of embryos available allows even an inexperienced researcher to perform hundreds of such manipulations per day. Transgenesis is quite effective as well. Finally, because the fate map of Xenopus embryos is stereotypical, simple targeted microinjections can reliably deliver reagents into specific tissues and cell types for gene manipulation or for imaging. Although yolk opacity can hinder deep imaging in intact embryos, almost any cell in the early embryo can be placed into organotypic culture, such that the cells of interest are directly apposed to the cover glass. Furthermore, live imaging techniques can be complemented with immunostaining and in situ hybridization approaches in fixed tissues. This protocol describes methods for labeling and high-magnification time-lapse imaging of cell biological and developmental processes in Xenopus embryos by confocal microscopy.
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