Immunohistochemistry (IHC)

All immunohistochemical (IHC) analyses were performed on paraffin-embedded tissues obtained from the primary tumor in the surgical specimen. For all IHC analyses the surgically resected specimens were fixed in 10% formalin and embedded in paraffin for routine pathological examination. We prepared and used 5-μm-thick paraffin sections cut from a paraffin block containing histological findings that were representative of the tumor. The procedure for IHC was previously described [27,28]. Antigen retrieval was performed in citrate buffer solution (pH 6.0). Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 15 min and all slides were heated to 95°C by exposure to microwave irradiation for 20 min and then cooled at room temperature (RT). Slides were washed in PBS and after a 1 h incubation at RT with the primary antibodies, the slides were incubated for 30 min with a labeled polymer EnVision TM+, Peroxidase–conjugated anti-Mouse or Rabbit (Dako, Tokyo, Japan). The chromogen used was 2% 3, 3^′-diaminobenzidine (DAB) in 50 mM Tris-buffer (pH 7.6) containing 0.3% hydrogen.