References

Label retaining cells (LRCs) in the skin were identified as previously described (S1). Briefly, four day old mice were injected daily for seven consecutive days with 50mg/kg body weight bromodeoxyuridine (BrdU) dissolved in phosphate buffered saline (PBS). Mice were sacrificed on day 80 to identify LRCs. Whole-mounts of mouse tail epidermis were prepared by peeling the tail skin and subsequently incubating the samples in PBS with 5 mM EDTA at 37 °C for 4 hours. Fixed epidermis were then washed with PBS, blocked in blocking buffer (5 % BSA in PBS), and then stained with anti-BrdU-FITC (Roche, 1: 50) overnight at 4 °C. A Leica SP1 Confocal system was used to obtain fluorescence images. Z-series of images were obtained throughout the hair follicle with a 20x, 0.7-numerical-aperture Plan-Apochromat objective and 3-dimensional reconstructions were analyzed quantitaitvely for BrdU positive cells using Imaris 5.0 software from Bitplane AG (Zurich, Switzerland). Assessment of the bulge CD34 population was analyzed using an anti-CD34 Ab (BD Pharmingen, 1: 200) incubated overnight at 4o C followed by a 2 hour incubation with a goat-anti-rat Ab conjugated with Alex Flour 488 (Molecular probes, 1: 200). Transiently amplifying cells were identifie