CONFOCAL LASER-SCANNING MICROSCOPY OF APOPTOSIS IN ORGANOGENESIS-STAGE MOUSE EMBRYOS

Confocal Laser scanning microscopy combined with a vital stain has bee n used to study apoptosis in organogenesis-stage mouse embryos. In ord er to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mous e embryos were harvested on gestation day 8 or 9 and stained with the vital lysosomal dye, LysoTracker Red. Following incubation in the stai n, embryos were fixed in 2% paraformaldehyde overnight, dehydrated hi a graded methanol series, and cleared in benzyl alcohol/benzyl benzoat e, The resulting embryo is almost transparent and retains specific Lys oTracker Red staining. The entire embryo can be optically sectioned an d reconstructed in three dimensions to reveal areas of dye staining. T o test this approach, the chemotherapeutic drug hydroxyurea was added to day 8 embryos in vitro to induce apoptosis. Our results demonstrate d specific regions undergoing programmed cell death in normal developm ent and increased apoptosis in embryos exposed to hydroxyurea. The obs erved patterns of LysoTracker Red staining correlate well with previou s studies of cell death using other lysosomotropic dyes such as Nile b lue 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 fo r the optical imaging of whole day 9 (similar to 22 somites) embryos t hat were greater than 500 microns thick in the Z-axis. Cytometry 33:34 8-354, 1998. (C) 1998 Wiley-Liss, Inc.dagger.