Anti p65 nf kb

Immunocytochemistry analysis was performed on coverslip-adherent cells by the labeled streptavidin biotin method using a LSAB kit (DAKO Japan, Kyoto, Japan). The samples were incubated with 0.1% Triton X-100 solution for 1 h, washed 3 times in PBS and treated for 40 min at room temperature with 10% BSA. In addition, coverslips were incubated overnight at 4 °C with the primary antibodies (anti-p53, anti-p65-NF-kB, anti-TGF-β1, diluted 1:400 and anti-Nrf2, diluted 1:300, Santa Cruz Biotechnology). Next, secondary antibody treatment was performed (2 h at room temperature), and horseradish peroxidase activity was visualized by treatment with H₂O₂ and 3,3′-diaminobenzidine (DAB) for 5 min. For the final step, the sections were weakly counterstained with Harry’s hematoxylin (Merck). For each case, negative controls were performed by omitting the primary antibody. Intensity and localization of immunoreactivities against the primary antibody used were examined on all coverslips using a photomicroscope (Olympus BX41, Olympus Optical Co., Ltd., Tokyo, Japan). For image analysis, color images of representative areas (400×) were digitally captured. For p53, p65-NF-kB and Nrf2 analyses, the percentage of cells with labeled nuclei were calculated.

Immunohistochemical staining was performed using anti-p-65-NF-kB (1:300), anti-MMP-9 (1:600) and anti-TIMP-1 (1:100) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), by the biotin–streptavidin–peroxidase method. Using the same point counting technique described above, we assessed cells positive for p65-NF-kB, MMP-9 and TIMP-1. Counting was performed in 20 fields of airway wall samples for each animal (5 airways per animal) at 1,000 magnification. Results were expressed as positive cells per area (10⁴μm²) [34 (link), 35 (link)]. All morphometric analysis was performed by two researchers blind to the genotype.