High-resolution imaging at the cellular and subcellular levels in flattened whole mounts of early zebrafish embryos
Abstract
We describe a rapid and sensitive method for high-resolution imaging at the cellular and subcellular levels in the whole-mount zebrafish embryo. The procedure involves fixing and staining the embryo, followed by deyolking and flattening it under a cover slip, to produce a planar mount that is 20 to 100 microns thick. Such a flattened whole mount allows imaging with a spatial resolution of approximately 500 nm in the x-y plane and does not require the use of embedding, sectioning, confocal microscopy, or computational deblurring procedures. We can resolve all individual nuclei and chromosome sets in the embryo, up to the late gastrula stage (10,000 cell stage). In addition, older embryos (through the segmentation stage) can also be examined, with the preservation of significant morphological detail. Because of its ability to resolve subcellular detail, the flattened whole-mount method can provide significant biological information beyond what can be obtained from conventional (three-dimensional) whole mounts. We have used the flattened whole-mount method to study subcellular events related to progression through the cell cycle or to apoptosis, in cells of the early zebrafish embryo. A specific DNA-binding dye (Hoechst 33258) or ...Continue Reading
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Apoptosis
Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis