Tissue samples were
first deparaffinized and rehydrated. Citrate
buffer (pH = 6.0) was used for antigen retrieval with microwave
heating. Endogenous peroxidase activity was blocked by%3
hydrogen peroxide in methanol. Then, NF-kB primary antibody
(1/100 dilution) (anti-NF-kB p65 antibody, Santa Cruz Biotechnol-
ogy, Texas, USA) was dropped and incubated overnight at 4 C.
After incubation, HRP secondary antibody kit was used as a
secondary antibody. AEC kit was used for chromogen. At last, all the
slides were counterstained with Mayers hematoxylin and mounted
with water based mounting medium. All the chemicals were
purchased from Labvision Corp. (Fremont, CA, USA). The immunoreactivity
of NF-kB was investigated by immunohistochemical
method.