All specimens were fixed in formalin and embedded in paraffin for examination. Sections (4 µm thick) were stained with H&E and analyzed by immunohistochemistry. The paraffin-embedded sections were deparaffinized and rehydrated, washed in distilled water, and then subjected to heat-mediated antigen retrieval. Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide in methanol for 15 min, followed by clearing in PBS for 5 min. The sections were blocked for 30 min with 3% normal horse serum diluted in PBS, blotted, and incubated with primary mouse anti-mouse proliferating cell nuclear antigen (PCNA, 1∶200 dilution) monoclonal antibodies in blocking serum for 4 h at room temperature. Thereafter, the slides were washed three times for 5 min each in PBS and incubated with biotinylated anti-mouse and anti-rabbit antibodies for 2 h. The slides were washed in PBS, followed by the addition of the avidin biotin peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA, USA). The slides were washed and the peroxidase reaction was developed with diaminobenzidine (DAB) and peroxide, followed by counterstaining with hematoxylin, mounting in aqua-mount, and evaluation under a light microscope (magnification×200; Olympus, Tokyo, Japan). A negative control was included in all experiments by omitting the primary antibody. For the detection of apoptotic cell death in the tumor tissues, the paraffin-embedded sections were incubated in a mixture of the labeling solution (540 µl) and enzyme solution (60 µl) for 1 h at 37°C, and then washed three times in 0.1 M PBS for 5 min each, according to the manufacturer’s instructions. Next, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min at 37°C. Finally, the sections were rinsed, mounted on slides, and cover-slipped for fluorescence microscopy (DAS microscope).