*2.15. Immunohistochemical Analysis*

Immunohistochemical analysis was performed as already described [50]. Tissues were fixed in 10% ( *w*/*v*) PBS-bu ffered formaldehyde and 7 μm sections were prepared from para ffin embedded tissues. After depara ffinization, endogenous peroxidase was quenched with 0.3% (*v*/*v*) hydrogen peroxide in 60% (*v*/*v*) methanol for 30 min. The sections were permeabilized with 0.1% ( *w*/*v*) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (*v*/*v*) normal goa<sup>t</sup> serum in PBS for 20 min. Endogenous biotin and avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin (DBA, Milan, Italy). Subsequently, the sections were incubated overnight with: anti-nitrotyrosine antibody (1:100; Millipore, Abingdon, UK) or anti-PARP antibody (1:100; Santa Cruz Biotechnology). Sections were washed with PBS and incubated with peroxidase-conjugated bovine anti-mouse IgG, secondary antibody (1:2000 Jackson Immuno Research, WestGrove, Pennsylvania, USA). Specific labeling was provided with a biotin-conjugated goa<sup>t</sup> anti-mouse IgG and avidin-biotin peroxidase complex (Vector Laboratories, Burlingame, California, USA). Images were collected using a Leica DM6 (Milan, Italy) microscope. The percentage area of immunoreactivity (described by the number of positive pixels) was reported as percent of total tissue area (red staining).
