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The percentual area fraction of live, degenerative and necrotic areas in the representative sections cut through the middle of the tumor were estimated by dividing the size of each tissue area by the total tumor section volume [ 27].
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The total mammary tumor section areas were measured using a special Macro-Stand device (support with Canon TV zoom lens V6×16/16-100 mm plus a Canon 58 mm close-up 240 lens connected to a CCD black-and-white video camera module with a Sony DC-777 camera unit) connected to a KS-300.
The percentage of collagen was quantified by calculating the ratio of blue staining (collagen) area in the total area of the tumor section using Imagescope analysis software [ 33].
To take into consideration the total tumor burden, H&E-stained sections were analyzed for total tumor burden in an independent cohort of mice killed after the fourth dose of cisplatin.
The ratio of the CD31-stained area to the total area of tumor sections from both treatment groups were analyzed, as well as the number of vessels in a viable tumor area.
In order to assess proliferation with Ki67, immunostained cells were counted throughout each tumor section and the total area of each section was then calculated to find the number of proliferating cells/mm.
For counting of necrotic cells, and activated caspase-3+ and cleaved PARP-1+ cells, tumors were bisected, and two images were captured from each tumor section to generate a total of 8 images from each tumor.
For the evaluation of the intratumoral vascular density, anti-CD-34 (1 200; Abcam, Cambridge, United Kingdom) immunohistochemistry, detecting human endothelial cells was performed for all tumors on a large section that included the total tumor.
MetaMorph software was used to analyze total tumor burden and osteoclast number in multiple sections from the femora and tibiae of the hind limbs.
Total tumor DNA was extracted from a 10 μm section from each biopsy, as previously described [ 19].
The total tumor volume is calculated as summed tumor area on all sections multiplied by width of the sections.
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