Immunohistochemical staining of nuclear factor (NF-κB /p65)
For immunohistochemical evaluation, histological sections were incubated at 60ºC overnight, and then de-waxed in xylene for 30 min. After rehydrating in a decreasing series of ethanol, sections were washed with distilled water and Phosphate buffered saline (PBS) for 10 min. Sections were then treated with 2% trypsin in 50 mM Tris buffer (pH 7.5) at 37ºC for 15 min and washed with PBS. Sections were delineated with a Dako pen (Dako, Glostrup, Denmark) and incubated in a solution of 3% H2O2 for 15 min to inhibit endogenous peroxidase activity. Then, sections were incubated with NF-kB/p65 monoclonal antibody. Immunohistochemistry was performed using the standard method (avidin biotin peroxidase) according to