Quantification of terminal end buds and proliferating cell nuclear antigen staining. (A) High-resolution composite digital light microscopic representative images of developing mammary glands at 5, 8, and 14 weeks from control, CYT-1 and CYT-2 mice were recorded using an Axio Imager.A.1 microscope with an AxioCam MRc5 camera and AxioVision 4.7 imaging software (Zeiss, Thornwood, NJ, USA). LN, lymph node. *End of the gland area, arrowheads point to the end of the terminal end buds (TEBs). Scale bars = 1,000 μm. (B) The number of TEBs in 8-week-old mammary gland whole mount slides was counted manually under magnification in transgenic CYT-1 and CYT-2 mice and their respective sibling controls. ****P <0.0001 by one-way analysis of variance (ANOVA) using Tukey’s post-ANOVA multivariate analysis. (C) Proliferating cell nuclear antigen (PCNA)-positive TEBs (TEBs with at least one luminal cell displaying positive PCNA nuclear staining) and total number of TEBs in thin mammary tissue sections of 8-week-old CYT-1 mice and sibling controls were counted, and expressed as percent positive for PCNA. (D) Percent PCNA-positive cells were calculated for each PCNA-positive TEB within a section of 8-week mammary gland in four mice in each control and CYT-1 and were averaged. *P <0.05 by unpaired t test. (E) Representative hematoxylin and eosin (HE) and PCNA stained images of TEBs in 8-week mammary glands. Scale bars = 50 μm.