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 (seeNote 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.
