Skeletal Examination by Alizarin Staining

A skeletal examination of fetuses is required in regulatory embryo–fetal development studies. This chapter describes a method of skeletal examination using alizarin staining. All fetuses are removed from the mother by caesarean section before birth. The fetuses are first examined externally. For larger species (rabbit and minipig), an internal examination can be performed on the fresh soft tissues by microdissection. For smaller species, such as the rat and mouse, half of each litter is fixed for internal soft tissue examination. The other half is used for skeletal examination. A rapid examination of the fresh soft tissues is performed before evisceration and fixation. The staining process takes several days. Following staining, all bones are examined from the head to the tail, in ventral and dorsal positions.