Anti 3 nitrotyrosine 3 nt

Testes were fixed immediately in 10% buffered formalin solution after harvesting and were embedded in paraffin and sectioned into 5 μm thick sections onto glass slides. The sections were processed for H&E staining and TUNEL staining, as previously described [6 (link)]. For TUNEL staining, apoptotic cells exhibited a brown nuclear stain as TUNEL positive cells and were counted manually under a microscope. Four sections from each testis were counted. On each section, 30 seminiferous tubule's cross sections from each testis were selected in the same pattern, moving each slide without repetitive counting in a blinded fashion [31 (link)]. Results were presented as TUNEL positive cells per 10³ cells. For IHC staining, the sections were incubated with anti-3-nitrotyrosine (3-NT, Millipore, Temecula, CA, USA, 1 : 100) overnight at 4°C. After washing with PBS, the sections were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA, 1 : 300 dilutions in PBS) for 2 hours at room temperature. The sections were then treated with peroxidase substrate 3,3′-diaminobenzidine in the developing system provided by Vector Laboratories (Burlingame, CA, USA) and counterstained with hematoxylin.