Recent studies demonstrate that carbohydrates synthesized by specific glycosyltransferases play important roles in the development of the central nervous system. Among these carbohydrates, polysialic acid is a unique glycan that modulates functions of the neural cell adhesion molecule (NCAM) by attenuating NCAM-mediated interaction between neural cells. During brain development, polysialic acid is synthesized in a specific spatiotemporal pattern by two polysialyltransferases, ST8SiaII and ST8SiaIV. To study in vivo the roles of polysialic acid synthesized by each respective enzyme, we generated ST8SiaII and ST8SiaIV knockout mice. Single knockout ST8SiaII or ST8SiaIV mice show polysialic acid expression patterns differing from wild type, and those patterns indicate different roles of each gene during neural development. In this chapter, we discuss methods used to analyze polysialyltransferase knockout mice using immunohistochemistry of brain and primary cultures of neurons.