Preparation of fixed Xenopus embryos for confocal imaging

Although live imaging of embryonic development is a powerful approach, it is often essential to use immunostaining or in situ hybridization to reveal structures or to localize proteins or gene expression patterns. For these approaches, fixed embryos must be used, and Xenopus offers many advantages. Xenopus is a vertebrate tetrapod closely related to mammals. The large size of their embryos makes them ideal for imaging patterns of gene expression during development. Also, individual Xenopus embryonic cells are larger than those of other vertebrate models, making them suitable for imaging cell morphology and subcellular processes. Xenopus embryos are amenable to simple manipulations of gene function, including knockdown and misexpression, and the large numbers of embryos produced allow even an inexperienced researcher to perform such manipulations on hundreds of embryos 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. To improve image quality for fixed specimens, the pigment of the Xenopus embryo can be removed by bleaching. Also, although Xenopus embryos are opaque because of the large amounts of yolk stored in each cell, they can easily be rendered transparent using a process called clearing. This protocol describes the methods for bleaching and clearing Xenopus embryos, as well as a simple procedure for vibratome sectioning. These approaches are effective for imaging embryos and cells following immunostaining or in situ hybridization.