Digital sight ri1

Mammary glands were collected at autopsy and fixed in 10% formalin for 24 hours. Subsequently, they were embedded in paraffin and sectioned at 4 µm thickness. Tissue sections were stained with haematoxylin and eosin (H & E) for histopathological analysis. For immunohistochemistry, slides were incubated overnight at 4°C with antibodies to 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) (1:100, N45.1; JaICA/GENOX Corporation, Baltimore, MD) or nitrotyrosine (1:250, MAB5404; Millipore, Billerica, MA). This was followed by incubation with biotinylated secondary antibody and avidin/biotin peroxidase complex for 30 minutes each at room temperature. The sections were then stained with 3’-diaminobenzamine substrate and counterstained with Modified Harris Haematoxylin. Mammary gland sections from all animals per treatment group were stained for the above listed markers. Representative images were taken randomly with Nikon Eclipse E800 fitted to Nikon Digital Sight Ri 1. The staining was quantified using an Aperio ScanScope (Vista, CA). Quantification was performed by randomly selecting five sections and counting over 10,000 cells per slide. Positive staining for nitrotyrosine was found in the cell cytosol. For 8-oxo-dG, positive staining was localized in the nuclei of cells.