Identifying Quiescent Stem Cells in Hair Follicles.

Hair follicle stem cells (HFSCs) are noted for their relative quiescence and therefore can be distinguished from other cells by their differential history of cell division. Replicating cells can be labeled by pulsing the animals repeatedly with 5-bromo-2'-deoxyuridine (BrdU) or tritiated thymidine ([ ³ H]TdR), thymidine analogs that get incorporated into DNA during DNA synthesis. Because dividing cells dilute the label after each cell division, frequently dividing cells will lose the label over time while slow cycling cells will retain the label and thus are termed label retaining cells (LRCs). [ ³ H]TdR can be visualized by autoradiography and BrdU can be detected by immunofluorescence with anti-BrdU antibodies. Alternatively, a well-established tet-regulatable transgenic mouse model can be used to express histone H2B-GFP in epithelial proliferative cells and their dilution and retention of the GFP signal can be followed. In this chapter, we detail the steps to perform BrdU pulse-chase and H2B-GFP pulse-chase experiments to identify quiescent cells in the hair follicle.