Immunohistochemistry Protocol for RUNX3 and Ki-67 Detection

Immunohistochemistry was performed with the indirect enzyme-labeled antibody method, as described previously (28 (link),29 (link)). For detection of RUNX3, mouse anti-human monoclonal (2B3) RUNX3 antibody (dilution 1:500; Abcam, Cambridge, CA, USA) was used. For detection of Ki-67, rabbit anti-human monoclonal (30 (link)–9 (link)) anti-Ki-67 antibody purchased from Roche Applied Science (Penzberg, Germany) was used. TMA sections were deparaffinized with toluene and rehydrated in graded alcohols. After autoclaved for 15 min at 120°C in 10 mM citrate buffer (pH 6.0) for antigen retrieval, endogenous peroxidase was inactivated with 0.3% hydrogen peroxide in methanol for 15 min. The sections were then pre-incubated with 500 µg/ml normal goat IgG dissolved in 1% BSA in PBS (pH 7.4) for 1 h, reacted with primary antibodies for 16 h, washed with 0.075% Brij 35 in PBS, and then incubated with HRP-conjugated goat anti-mouse/rabbit (RUNX3/Ki-67) in 1% BSA in PBS for 1 h. After washing with 0.075% Brij 35 in PBS, the sites of HRP were visualized with DAB and H₂O₂. As a negative control, some sections were reacted with normal mouse IgG instead of the specific antibodies. The stained slides were analyzed under a laser scanning microscope (LSM 5 PASCAL; Carl Zeiss AG, Oberkochen, Germany).