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Keywords:

  • confocal laser scanning microscopy;
  • apoptosis;
  • LysoTracker Red;
  • hydroxyurea;
  • embryos;
  • clearing;
  • benzyl alcohol;
  • benzyl benzoate;
  • phagocytosis

Abstract

Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mouse embryos were harvested on gestation day 8 or 9 and stained with the vital lysosomal dye, LysoTracker Red. Following incubation in the stain, embryos were fixed in 2% paraformaldehyde overnight, dehydrated in a graded methanol series, and cleared in benzyl alcohol/ benzyl benzoate. The resulting embryo is almost transparent and retains specific LysoTracker Red staining. The entire embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. To test this approach, the chemotherapeutic drug hydroxyurea was added to day 8 embryos in vitro to induce apoptosis. Our results demonstrated specific regions undergoing programmed cell death in normal development and increased apoptosis in embryos exposed to hydroxyurea. The observed patterns of LysoTracker Red staining correlate well with previous studies of cell death using other lysosomotropic dyes such as Nile blue sulfate, acridine orange, or neutral red. LysoTracker Red has the advantages of being aldehyde-fixable and highly fluorescent (bleaching was not observed even after multiple scans). This procedure allows for the optical imaging of whole day 9 (≈22 somites) embryos that were greater than 500 microns thick in the Z-axis. Cytometry 33:348–354, 1998. Published 1998 Wiley-Liss, Inc.1