Immunohistochemical Analysis of Tumor Markers

Immunohistochemical analysis was performed as described previously [26 (link)]. Tumor tissue sections were incubated in xylene and passed through series of graded alcohols followed by antigen retrieval using the antigen retrieval solution (Vector Labs, Burlingame, CA). Tissue sections were incubated in 3% H₂O₂ solution for 20 min and then subjected to blocking using the Vector Lab Blocking Kit. Tissue sections were incubated overnight with primary antibodies of Ki-67 (1:100), Cox-2 (1:50), and IL-1 beta (1:100) and then with secondary antibodies. Immunoreactivity was visualized by using the DAB substrate and counterstained with hematoxylin (Vector Lab, Inc.). The proliferative index was calculated as percentage of Ki-67-positive cells in five randomly selected microscopic fields. TUNEL analysis was performed using the In situ Cell Death Detection Kit according to manufacturer's protocol and DAPI was used to visualize the nuclei. Percentage of apoptotic cells were calculated from five randomly selected microscopic fields in each group.