Confocal Laser Scanning Microscopy Morphology and Apoptosis in Organogenesis-Stage Mouse Embryos

In our efforts to use confocal laser scanning microscopy for study of organogenesisstage rodent embryos, we have developed fixation and clearing methods to allow optical sectioning through embryos with thickness approaching 1 mm (z -axis). We have combined fixation and clearing methods with fluorochrome staining for several purposes. In this chapter we present two methods; first, clearing with methyl salicylate (oil of wintergreen) and staining with Nile blue sulfate (NBS) (not used as a vital dye for this protocol) for general morphological assessment, and second, staining live embryos with the vital stain LysoTracker� Red (LT), followed by fixation and clearing with benzyl alcohol∶benzyl benzoate (BABB) to visualize areas of apoptosis (see Note 1 , ref. 1 ). With both protocols, an entire organogenesis-stage rodent embryo can be optically sectioned and reconstructed in three dimensions (3-D) to reveal areas of dye staining.