Rabbit anti human nf κb p65 primary antibody

NF-κB is a protein complex, which controls transcription of DNA. NF-κB is involved in cellular responses to stimuli such as stress, cytokines and free radicals. Therefore, our aim for using NF-κB staining method was to detect possible pathologies about oxidative stress in liver tissue. Immunohistochemical staining was performed on 7 μm thick paraffin-embedded biopsies. Immunohistochemical staining for NF-κB protein was performed by an automated method on the VENTANA BenchMark GX System (Ventana Medical Systems, Inc.) with an ultraView Universal DAB Detection Kit. The antigenic determinant sites for NF-κB were unmasked in citrate buffer with steam for 60 minutes, after deparaffinization step. The primary antibody used for NF-κB staining, a rabbit anti-human NF-κB/p65 primary antibody (Santa Cruz, CA) was used at a dilution of 1:50 for 32 minutes at 37°C. Then, the slides were incubated with the diluted antibody, followed by application of ultraView Universal DAB Detection Kit (Ventana Medical Systems, Inc.). DAB was used as a chromogenic and hematoxylin as a counter stain.

Immunohistochemistry by NF-κB-p65 and TUNNEL staining in paraffin sections was performed as follows: the sections were deparaffinized and treated with proteinase K solution (20 μg/mL in PBS) for 15 minutes at room temperature. Subsequently, the sections were washed in distilled water and immersed in 3% hydrogen peroxide for 15 minutes. After several washes with PBS (50 mM sodium phosphate and 200 mM NaCl at pH 7.4), the sections were immersed in an equilibration buffer at room temperature for 20 minutes. Some sections were incubated with rabbit anti-human NF-κB/p65 primary antibody (1:50, Santa Cruz Biotechnology, sc-109) at 37°C for one hour in a humidified chamber to detect immune and inflammatory responses. Others were incubated with terminal deoxynucleotidyl transferase and biotinylated dNTP (Life Technologies, Inc.) at 37°C for one hour in a humidified chamber in order to detect DNA breaks. Then, the reaction was stopped by immersion in a stop/wash buffer. After several washes, all sections were incubated in anti-digoxigenin-peroxidase for 30 minutes at room temperature. The reaction was revealed with 0.06% 3, 3-diaminobenzidine tetrahydrochloride (Sigma Chemical, St. Louis, MO) in PBS for three to six minutes, and the sections were counterstained with Mayer’s hematoxylin. The sections were examined and photographed under a light microscope (Olympus BH-40).