Fig. 4. IL-4/IL-13 expression is reduced in Stat6^-/- mammary glands and these cytokines are required for mammary gland development. (A) Quantitative real-time-PCR was performed using specific primers for Il4, Il13, Il5, Il12a and Gata3 on wild-type (WT) and Stat6^-/- glands at day 5 and 15 of gestation. Error bar represents s.d. of three samples and is representative of at least two experiments. (B-D) Whole mounts (left panels) and H&E-stained sections (right panels) of inguinal mammary glands from wild-type (strain matched) and IL-4^-/-/IL-13^-/- mice at day 5 (B), 10 (C) and 15 (D) of gestation. (E) The number of alveoli was scored in H&E images from wild-type and IL-4^-/-/IL-13^-/- mice at 40x magnification and the area of the fat pad was measured using ImageJ software. The number of alveoli/mm² was calculated and the percentage relative to wild-type was plotted. (F) Immunoblots for phosphorylated (p)Stat6 and Stat6 were performed on wild-type and IL-4^-/-/13^-/- mice at day 5 of gestation. The density of the bands was quantified using ImageJ software and the ratio of pStat6 to Stat6 plotted on a histogram.