Analysis of cell size and proliferation in early mammary morphogenesis. (A) Optical sections (planar and sagittal views) of confocal microscopy images from sparsely labeled R26-CreERT/tdTomato reporter embryos at placode (E11.5), hillock (E12.5), and bulb (E13.5) stages showing cytoplasmic expression of the reporter (tdTomato, red). Samples were stained with epithelial marker EpCAM (white) and nuclear marker Hoechst (cyan). (B) Optical sections (planar and sagittal views) of confocal images from transgenic Fucci cell cycle mouse model at placode (E11.5), hillock (E12.5), and bulb (E13.5) stages. Epithelial cells and nuclei have been stained with EpCAM (white) and Hoechst (cyan), respectively. The majority of the MECs are nonproliferative (G1/G0 phase, red), and only rare proliferative cells (S/G2/M phases, green) could be observed in the mammary bud. (C) Optical sections (planar and sagittal views) of confocal images of the mammary gland area from wild-type embryos at mammary gland placode (E11.5), hillock (E12.5), and bulb (E13.5) stages. Embryos were collected 2 h after EdU injection followed by whole-mount staining of EdU (S phase, green), epithelial marker EpCAM (white), and nuclear marker Hoechst (cyan). (A–C) Dashed line marks the epithelial-mesenchymal border, and arrowheads indicate the plane of the optical section. Scale bar, 50 µm.