At this time the field of view, focus and f stop were adjusted. Afterwards, the chamber door was closed to exclude room light. We allowed 5 minutes for (-)-Nutlin-3 the integration of the ICCD camera before images were acquired. Luciferase assay To measure luciferase reporter gene expression in doxy cycline induced and un induced mammary glands of double transgenic mice, all 5 mammary glands were dis sected, rinsed in PBS and tissues were homogenized in Reporter lysis buffer. Insoluble tissue lysates were removed by centrifugation at 4 C for 5 minutes. Luciferase activity was measured using 10ul of protein lysate, the Luciferase assay kit and a Berthold luminometer. The luciferase readings were normalized to total protein concentration.

Edu proliferation assay For assessment of cell proliferation within the mammary gland, the fourth mammary glands from doxycycline induced and un induced double transgenic mice were harvested at 10. 5 days postcoitus and 5um thick sections were embedded in paraffin. Cell proliferation was detected using incorporation of 5 ethynyl 2 deox yuridine with the Click iT EdU Cell Proliferation Assay Kit, following the manufacturers instructions. EdU that had been incorpo rated into newly synthesized DNA was detected by Alexa Fluor 594 azide and cell nuclei were stained with Hoechst 33342. The proportion of nucleated cells incorporating EdU was determined by fluorescence microscopy. Fifteen random 20�� fields were taken from each group of litter matched doxycycline induced and un induced double transgenic mice.

The proliferat ing cells were quantified and normalized to the total cell number in each field. Whole mount analysis Whole mount preparation of mammary glands was per formed at various time points as previously described. Briefly, mammary glands were removed from doxy cycline induced and un induced double transgenic mice and fixed overnight in acetic acid ethanol solution. AV-951 Fixed mammary glands were then dehydrated using 70% ethanol for 30 minutes and stained overnight with Car mine stain. The mammary glands were then destained, dehydrated through a series of washes in 70%, 95% and 100% ethanol for 30 minutes each and defatted in xylene. Histological staining and immunohistochemistry The third mammary glands from doxycycline induced and un induced double transgenic mice were fixed and embedded in paraffin.

Five micrometer thick sections were deparaffinized with xylene and stained with hema toxylin and eosin or used for immunohistochem istry. For IHC, antigen retrieval was performed by treating deparaffinized sections with sodium citrate buf fer at 95 C for 20 minutes. The sections were then blocked for one hour with serum followed by an additional 10 minute blocking with hydrogen peroxide. Sections were incubated MEK162 structure with rabbit anti TBX3 and rabbit anti NF BIB antibodies overnight at 4 C. The following day, sections were washed in PBS and incubated with biotinylated goat anti rabbit IgG.