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Cold Spring Harb Protoc. 2013 Apr 1;2013(4):362-5. doi: 10.1101/pdb.prot073874.

Microsurgical approaches to isolate tissues from Xenopus embryos for imaging morphogenesis.

Abstract

To understand dynamic cell and tissue mechanics during Xenopus laevis morphogenesis requires the use of live reporters of cell architecture as well as reporters for the location and activity of proteins in cells within a multicellular tissue. Fluorescently tagged proteins are some of the most useful reporters for live cell work. Once the desired expression pattern of fluorescent reporters has been found, the next step is preparing samples of tissue explants to bring subcellular structures and protein dynamics within the range of high-numerical aperture (NA) objectives. Visualization of dynamic living structures like the cytoskeleton requires the use of high-resolution confocal microscopes outfitted with oil-immersion, high-NA objectives. These objectives, typically 40×, 63×, and 100×, have very short working distances (i.e., samples must be brought within 150 μm of the coverslip, but perform optimally only when samples are <25 μm from the coverslip). Thus, to visualize protein dynamics and follow cell behaviors during morphogenetic events, tissues must be isolated by microsurgery and cultured on the glass surface of custom-made culture chambers. To visualize multicellular tissues from Xenopus embryos, we microsurgically dissect tissues from the desired part of the embryo using fine tools made from human hair. In this protocol, we discuss tools, microsurgery technique, and specific surgical manipulations to isolate important tissues from the gastrulating embryo.

PMID:
23547158
PMCID:
PMC4064588
DOI:
10.1101/pdb.prot073874
[Indexed for MEDLINE]
Free PMC Article

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