Histological Analysis of Mammary Tumors

Mammary tumors were frozen in OCT or fixed for 2 hours in 4% paraformaldehyde, sectioned and stained with hematoxylin and eosin as described previously [28 (link)]. For frozen sections, 5μm thick sections were fixed in acetone for 5′ at room temperature and stained with the following antibodies: K8 (1:200, Developmental Studies Hybridoma Bank, TROMA-1), CD45 (1:100, BD Biosciences, 550539), ADAM17 (1:200, Abcam, ab2051), F4/80 (1:100, BioRad, MCA49RT) and Cox-2 (1:200, Santa Cruz Biotechnology, sc-1747). For staining of paraffin-embedded tissues, the following antibodies and conditions were used. F4/80 (1:100; no antigen retrieval, MCA49RT, BioRad) and BrdU (1:300, sodium citrate antigen retrieval, Abcam, ab6326). For quantification of staining, total numbers of positive cells were counted in at least five 40x images taken from a minimum of three tumors per genotype.