Document Detail


High-resolution imaging at the cellular and subcellular levels in flattened whole mounts of early zebrafish embryos.
MedLine Citation:
PMID:  9551178     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 an antibody against a chromosomal protein (histone H1) was used to stain the nuclei of individual cells in the embryo. This allowed us to determine the spatial positions of all the individual cells, and also their stages in the cell cycle. A terminal transferase (TUNEL) assay was used to detect apoptotic cells. This combination of specific stains allowed us to study the behaviors of groups of cells in situ, within the developing zebrafish embryo.
Authors:
T D Yager; R Ikegami; A K Rivera-Bennetts; C Zhao; D Brooker
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemistry and cell biology = Biochimie et biologie cellulaire     Volume:  75     ISSN:  0829-8211     ISO Abbreviation:  Biochem. Cell Biol.     Publication Date:  1997  
Date Detail:
Created Date:  1998-05-27     Completed Date:  1998-05-27     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8606068     Medline TA:  Biochem Cell Biol     Country:  CANADA    
Other Details:
Languages:  eng     Pagination:  535-50     Citation Subset:  IM    
Affiliation:
Division of Developmental Biology, Hospital for Sick Children, Toronto, ON, Canada. yager@sickkids.on.ca
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Cycle
Cell Nucleus
Chromosomes
DNA
DNA Fragmentation
Embryo, Nonmammalian / cytology
Fluorescent Dyes
Histones
Immunoenzyme Techniques
Microscopy, Fluorescence / methods*
Sensitivity and Specificity
Staining and Labeling / methods*
Zebrafish / embryology*
Chemical
Reg. No./Substance:
0/Fluorescent Dyes; 0/Histones; 9007-49-2/DNA

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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