Fig. 4. Reduced epithelial proliferation in Dgat1^–/– mammary glands during pregnancy. (A) Bromodeoxyuridine (BrdU) immunostaining. Pregnant (P15) mice were injected with BrdU 2 hours before mammary glands were removed. Tissues were fixed, embedded and sectioned, and proliferating nuclei were detected by BrdU immunostaining (dark brown staining). (B) Quantification of BrdU-positive nuclei. Epithelial areas were calculated using images of histological sections (see Materials and methods), and positive nuclei from five random fields were counted and expressed per unit of area (^*P<0.05, n=4 mice per genotype). The expression of BrdU-positive nuclei as a percentage of total epithelial nuclei showed a similar reduction in proliferating nuclei (9.76±1.02% for Dgat1^–/– versus 17.82±0.3% for wild type, P=0.002, n=3). (C) Similar levels of apoptosis were seen in mammary glands of wild-type and Dgat1^–/– mice at P18. Apoptotic epithelial cells were detected by TUNEL staining and were counted in 10 random fields. n=5 mice per genotype.