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MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES, EMBRYOS AND FETAL LIMBS USING CONFOCAL MICROSCOPY
Citation:
Zucker, R M. MAMMALIAN APOPTOSIS IN WHOLE NEONATAL OVARIES, EMBRYOS AND FETAL LIMBS USING CONFOCAL MICROSCOPY. Presented at Microscopy & Microanalysis, Long Beach, CA, August 04 - 09, 2001.
Description:
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. The background staining with LT is also sufficient to visualize morphological structures. After LT staining for 30min-3hours, the tissues were fixed with either 2% glutaraldehyde or 4% paraformaldehyde. The tissues were then dehydrated with MEOH and cleared with benzyl alcohol/benzyl aldehyde. Images were acquired using high NA 5-20x objectives and 568 nm laser excitation.
In the limb, the LT concentrated between the digits in a normal 15th day rat and in the proliferating progress zone of a 14th day rat after 5-FU treatment. LT correlates and co-labels with other cell death probes (i.e. TUNEL, annexin, neutral red, acridine orange, Nile blue, pyknotic nuclei) in the limb, strongly suggestive that it is also measuring cell death. In the embryo, LT showed increase labeling of the otic pit (ear), brachial arches, and optic cup (eye) during normal development and after treatment in vivo or in vitro with various toxicants. Ovaries derived from 10-45 day old mice/rats, contained follicles that showed gross morphologic changes typical of atresia were also characterized by intense LT staining of granulosa cells. The observation of LT in embryos, limbs and ovaries suggests that LT is a fluorescent probe that measures apoptosis in solid tissues.