Immunohistochemical Quantification of Biomarkers

Consecutive 4-μm thick sections were immunohistochemically stained using the immunoperoxidase technique described previously [45 (link)], with the primary antibodies listed in Table 5 and appropriate secondary antibodies (Medical and Biological Laboratories, Nagoya, Japan) (all 0.2 µg/mL). The tissue sections were then color-developed with diamine benzidine hydrochloride (Dako, Glostrup, Denmark) and counterstained with Meyer’s hematoxylin (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). Staining indices were evaluated by examining 1000 epithelial cells, and the frequency of positive nuclear staining was determined. For p53 and NS, an index of more than 10% was considered to represent a “positive case”. TERT evaluation was performed by assessing 100 cells at the bottom of the glands, and a positive cell number per 20 cells was considered to represent TERT expression.