Pubertal Ductal Morphogenesis: Isolation and Transcriptome Analysis of the Terminal End Bud

The terminal end bud (TEB) is the growing part of the ductal mammary epithelium during puberty, enabling the formation of a primary epithelial network. These highly proliferative bulbous end structures that drive the ductal expansion into the mammary fat pad comprise an outer cap cell layer, containing the progenitor cells of the ductal myoepithelium, and the body cells, which form the luminal epithelium. As TEB make up only a very small part of the whole mammary tissue, TEB-associated factors can be easily missed when whole-tissue sections are being analyzed. Here we describe a method to enzymatically separate TEB and ducts, respectively, from the surrounding stroma of pubertal mice in order to perform transcriptomic or proteomic analysis on the isolated structures and identify potential novel regulators of epithelial outgrowth, or to allow further cell culturing. This approach has previously allowed us to identify novel TEB-associated proteins, including several axonal guidance proteins. We further include protocols for the culturing of isolated TEB, processing of mammary tissue into paraffin and immunohistochemical/fluorescent staining for verification, and localization of protein expression in the mammary tissue at different developmental time points.