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