Polyclonal rabbit anti rat nf κb p65

After being dried for 45 minutes, paraffin sections were dewaxed in 2 changes of xylene for 15 and 20 minutes each, followed by a descending ethanol series and antigen retrieval in ethylene diamine tetra-acetic acid. The sections were incubated in 3% hydrogen peroxide for 15 min in a humidistat box at room temperature and rinsed in phosphate balanced solution (PBS) for 5 min⁡× 3. After incubating overnight at 4°C with polyclonal rabbit anti-rat NF-κB p65 (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA), the sections kept in the humidistat box were warmed to 37°C in an incubator for 45 minutes and then incubated with secondary antibodies (Immunohistochemistry Kit, Diagnostic BioSystems, Pleasanton, CA, USA) at 37°C for 30 min. After being washed with PBS for 5 min⁡× 3, the sections were made visible by using 3,3-diaminobenzidine, terminated on distilled water, and, subsequently, counterstained with hematoxylin for 1 minute. Finally, the slides were differentiated in 1% acid alcohol, blued in 1% ammonia water, dehydrated in graded concentrations of ethanol, cleared in 2 changes of xylene for 10 minutes each, and mounted with neutral gum. The sections were examined and photographed with a Zeiss Primo Star, light microscope at ×400.

Diethylnitrosamine (DEN) and CCl₄ were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Magnolia fargesii with a voucher specimen (Lee, J. & M. S. Yang 021) was obtained from Gyeongsang National University, South Korea. LBDE with molecular weight 446.5 g/mol was isolated as described previously [17 (link)]. Polyclonal rabbit anti-rat NF-κBP65 and I-κBα were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rat secondary antibody labelled with horseradish peroxidase was taken from Cell Signalling (Beverly, MA). The Nitrocellulose membrane was used for the transfer proteins (Bio-Rad, CA). The visualisation of proteins was done with ECL and detection kit (Amersham Pharmacia Biotech, UK). A bicinchonic acid (BCA) protein assay kit for protein quantification was provided by Pierce Biotechnology, Rockford.