Mammalian Apoptosis in Whole Neonatal Ovaries, Embryos and Fetal Limbs Using Confocal Microscopy

Abstract

The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional sectioning approaches. However, the imaging of such whole-mounts presents technical problems of its own. One of the major problems with using a confocal microscope to image whole organs and embryos is the depth of penetration of the laser light into the tissue. We have optimized the confocal microscope performance and developed a sample technique that increases the optical resolution of the system.

CLSM has been used to study cellular death (apoptosis) during GD 8-12 normal rat/mouse embryonic development, neonatal ovarian development (PD10-45) and fetal limb development (GD 11-15). LysoTracker Red (LT) was incorporated into the tissue prior to laser excitation. It is aldehyde fixable stain that concentrates into acidic structures or into cells that have high lysosomal activity.