Human macrophages enhance humanization of the murine mammary gland. Murine mammary glands were humanized +/- the addition of primary human macrophages and exogenous estrogen supplementation. Humanization was repeated in two independent experiments using different patient/donor-derived normal breast fibroblasts and macrophages between experiments. The primary breast epithelial cells and organoids used for all experiments originated from the same patient. A minimum of ten glands from each treatment group was used for analyses. (A) Schema representing steps of procedure. (B) Total and humanized areas were determined after two months of growth by histological examination of H&E-stained tissue, imaged with the Zeiss SteREO Discovery V12 and calculated using Axiovision V4.8 software. (C) H&E-stained humanized histosections. Images were captured at 40× (left) and 20× (right) magnification. (D) Fluorescence in situ hybridization (FISH) analysis of humanized mammary glands. Paraffin-embedded histosections of humanized mammary glands were subjected to FISH analysis to confirm the species of origin for the identified regions of humanization. Human X chromosome probe was labeled with biotin-16-dUTP (green). Slides were counterstained with diamidino-2-phenylindole (DAPI, blue) and mounted with antifade. Analysis was performed using the Zeiss Axioplan 2 fluorescence microscope coupled with a CCD camera and images were captured with FISHview 4.5 software at 100× magnification. (E) Data represent percent humanization ± standard error (SE) of two independent experiments. A minimum of ten glands per treatment group were measured. *P < 0.05. FB, fibroblast conditioned media; ϕ, macrophage conditioned media; E2, estrogen treatment.