TROP2 Defines Branch Formation in Fetal Kidney Figure 2. Characteristics of ureteric bud cells in the developing kidney. (A–B) Immunostaining of E11.5 (A) and E14.5 (B) kidney with anti- EpCAM (green) and anti-TROP2 (red) Abs and nuclei (blue). (A) Arrowheads indicate the Wolffian duct and the arrow indicates the ureteric bud. Higher magnification images of the boxed regions are shown underneath. In the lower figure of A, the ureteric bud grows toward the bottom and the yellow arrowhead indicates the tip of the ureteric bud. Scale bars = 50 mm. (B) The dotted line delineates the periphery of the kidney. Scale bars = 100 mm. (C) Flow cytometry of E12.5 and E14.5 kidney cells using anti-EpCAM and anti-TROP2 Abs. The boxed regions and the dotted boxed regions show TROP2+ and TROP22 cell populations, respectively. (D) Fractionation of EpCAM+ cells in E14.5 kidney using anti-EpCAM and anti-TROP2 Abs. The upper, middle and lower boxed regions in the panel indicate the EpCAM+TROP2high, EpCAM+TROP2low and EpCAM+TROP2nega cell population, respectively. (E) Quantitative RT-PCR analysis of marker genes in freshly isolated E14.5 EpCAM+TROP2nega(2), EpCAM+TROP2low(+) and EpCAM+TROP2high(++) cells. The relative gene expression of marker/GAPDH is shown. Error bars are s.d. doi:10.1371/journal.pone.0028607.g002 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28607