Pregnancy induces proliferation in both the HR+ and HR- cells. (A) Representative images of carmine-alum-stained whole mounts of mammary glands from virgin, 3-day and 7-day pregnant FVB/N mice. Scale bar, 1 mm. (B) Confocal immunofluorescence of mammary gland sections from virgin, 3-day and 7-day pregnant mice stained for the proliferation marker 5-ethynyl-2’-deoxyuridine (EdU, green), Estrogen Receptor (ER, red) and the luminal cell marker cytokeratin-8 (CK8, blue). Scale bar, 10 μm. The exposure time for the ER signal was increased in pregnant samples to allow robust identification of ER+ cells (see Additional file 3 for images with a fixed exposure time). (C) Quantification of the proportion of luminal cells that is proliferating, separated by cell type based on ER expression. ER+ and ER- luminal cells start proliferating at day 3 of pregnancy, by day 7 the majority of proliferating luminal cells are ER-. Bars represent individual animals.