Animals were supplemented with 17β-estradiol at a total dose of 0.72 mg/pellet 7 days before inoculation with BT-474 tumor cells. When tumor volume reached 200 mm3 and 500 mm3, tumors were excised from the body and embedded in OCT medium and snap frozen after MR imaging. Animals were euthanized using CO2 inhalation after tumor excision. Twenty micrometer sections were cut and fixed in 10% paraformaldehyde solution followed by either hematoxylin or eosin (H&E) for detection of tumor necrosis or immunostaining for endothelial cell density. For immunostaining of endothelial cell density, CD 31 antibody (rat-anti-mouse, AbD Serotec, USA) was used as the primary antibody. Primary antibody was detected by sequential incubation with secondary antibody (goat anti-rat IgG conjugated to Cy3, Life Technologies, NY, USA). Images were captured and processed with an epifluorescence microscope (Nikon E600 Upright Microscope, NY, USA) and ImageJ software (NIH, USA). Five mice were used in each group.
The fractional area of tumor necrosis was determined from digital images of H&E stained whole tumor sections using Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, USA). The whole tumor and regions of necrosis were manually delineated, and the necrotic fraction was calculated as the ratio of the necrotic area to the whole tumor area.
The fractional area of tumor necrosis was determined from digital images of H&E stained whole tumor sections using Adobe Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, USA). The whole tumor and regions of necrosis were manually delineated, and the necrotic fraction was calculated as the ratio of the necrotic area to the whole tumor area.