Quantitative analysis of mammary ductal elongation and branching. Mammary gland #4 was isolated from 5-week-old, 8-week-old and 14-week-old (5w, 8w and 14w) CYT-1 (red circle) and CYT-2 (red triangle) ERBB4 transgenic mice and their respective transgene negative sibling controls (blue circles and triangles), and was processed for whole mount analysis using Carmine Alum stain. (A) Stained whole mounts were photographed under a dissection microscope with a SPOT 11.2 Color Mosaic camera at 10× magnification using SPOT advanced software 4.0.9 (Diagnostic Instruments Inc; Sterling Heights, MI, USA). Ductal growth and branching were measured using ImageJ software (National Institute of Health, Bethesda, Maryland, USA). (B) For ductal branching, branch points were counted manually in three random unit areas using ImageJ software and averaged for each mouse. Data points indicate branch points ± standard error of the mean (SEM) in each group. (C) Ductal elongation/growth was calculated as the percentage of the distance from the lymph node to the farthest point of the longest duct relative to the distance to the farthest limit of the mammary fat pad. Data points indicate percent growth ± SEM in each group. ***P <0.001, **P <0.01, *P <0.05 by one-way analysis of variance (ANOVA) using Newman–Keuls post-ANOVA multivariate analysis.