(a) Schematic diagram showing the rationale for quantitative in vivo single-BEC tracing. (b) Experimental design. (c) Upon administration of a very low dosage of tamoxifen (0.25 mg/kg body weight), liver samples were stained with anti-CK19 antibody and Hoechst33342. BEC labeling was introduced at the single-cell level (white arrows). A 3D image and an optical section corresponding to the same visual field are shown in the left and right panels, respectively (scale bars, 100 μm). (d) Quantification of the BEC-labeling efficiency after the low-dosage tamoxifen injection. For FACS analysis, successive gates were applied for DAPI-, FSC/SSC, pulse width and EpCAM+ (not shown). A representative plot pattern for 4 biological replicates is shown. (e and f) 3D images of labeled colonies after 6 weeks of TAA injury. Thick sections were stained with anti-CK19 antibody and 3D images were acquired with tdTomato+ colonies (white arrows) using confocal microscopy. The data are shown as maximum intensity projections. (e) Duct compartment around the PV area. (f) Peripheral ductule compartment around the CV area (scale bars, 100 μm). (g) Distribution of the quantified colony size at TAA 6 weeks (n = 5 mice, mean ± SD). The colonies were classified into two categories (duct and peripheral ductule) as described in the 'Materials and methods' section. (h) Relative numbers of colonies categorized by colony size as depicted in the legend to the right (left stacked bar chart), and the relative contribution of cell amounts from each colony category (right stacked bar chart) (calculated as follows: 100 x (sum of the cell numbers in a colony size)/(sum of all the counted cell numbers)). (i) Scatter plot of the colony size distribution over time. Data from five mice were pooled for each time point (total colony numbers counted were 257, 272, 304, 307 and 310 for the 0, 2, 4, 6 and 8 week samples, respectively). Horizontal lines show the mean of colony size. Images shown in panels (c), (e), and (f) are representative data for at least 5 biological replicates.