Histostain plus bulk kit

For immunohistochemistry, sections were incubated with H₂O₂ (10%) for 30 min to eliminate endogenous peroxidase activity and blocked with 10% normal goat serum for 1 h at room temperature. Subsequently, sections were incubated in primary antibodies, Ki-67 (Bioss, Inc., Massachusetts, USA, 1/100), iNOS (Santa Cruz Biotechnology, Inc., Texas, USA, 1/100) for 24 h at 4 °C. Antibody detection was performed with the Histostain-Plus Bulk kit (Bioss, Inc., Massachusetts, USA) against rabbit IgG, and 3,3’ diaminobenzidine (DAB) was used to visualize the final product. All sections were washed in PBS and photographed. The slides used in immunohistochemical evaluation were selected at random by three blind observers. Observers examined Ki-67 and iNOS stained slides, eight per both, from each experimental group. One hundred cells were counted at 40× magnification (total magnification 400×). The number of cells that reacted positively was noted, while the latter score was obtained by taking the average. Based on this, immunohistochemical positivity was evaluated as follows: Score 0 (–): 0–5 cells with positive reactions, Score 1 (+): 5–15 cells with positive reactions, Score 2 (++): 15–25 cells with positive reactions, Score 3 (++): 26 and more cells with positive reactions.