Cold Spring Harb Protoc 2013 Apr 01;20134:366-9. doi: 10.1101/pdb.prot073882.

Assembly of chambers for stable long-term imaging of live Xenopus tissue.

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Understanding the molecular mechanisms and cellular mechanics that drive morphogenesis in Xenopus laevis embryos requires high-resolution quantitative imaging of cell movement and protein dynamics within multicellular tissues. To perform long-term tissue culture and stable imaging of live Xenopus tissue, we have developed a simple acrylic chamber that can be sealed and reused. The explants can be cultured on an adhesive substrate or within a nonadhesive microenvironment. We have developed a method for sandwiching isolated marginal zone (MZ) explants between thin agarose sheets. To manipulate the stiffness of the substrate or to measure cell traction generated by cells in an intact tissue, a fibronectin-conjugated polyacrylamide gel (FN-PAG) substrate can be used. The mechanical properties of this substrate can be easily modulated by varying the ratio of acrylamide to bis-acrylamide and the transparent nature of the PAG can allow observation of intracellular dynamics through the gel. Cell tractions are detected by following movements of fluorescent beads embedded in the gel.

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Species referenced: Xenopus laevis
Genes referenced: bag3 epha4 epha7 fn1

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