Immunohistochemical analysis was performed with a Dako Envision+Mouse/Rabbit Peroxidase Detection System (Dako Cytomation). Antigen retrieval was performed by pressure cooker heating in citrate buffer (pH 6.0) for 5 min. Peroxidase activity was blocked with 3% H2O2‐methanol for 10 min. Sections were incubated with a mouse monoclonal anti‐KHDRBS3 (SLM‐2) F‐3 antibody (1:200; Santa Cruz Biotechnology, Inc) for 1 h at room temperature, followed by incubation with Envision+anti‐mouse peroxidase for 1 h. For color reactions, sections were incubated with DAB Substrate‐Chromogen Solution (Dako Cytomation) for 5 min. Sections were counterstained with 0.1% hematoxylin. Reactions lacking a primary antibody were used as negative controls.
Two surgical pathologists (NS and DT) independently measured the ratio of positivity without knowledge of the clinical and pathological parameters or outcome of the patients. When >50% of tumor cells were stained, immunostaining was considered to be positive for KHDRBS3 in reference to the median value of the positivity. Inter‐observer differences were resolved by consensus review at a double‐headed microscope after independent reviews.
Two surgical pathologists (NS and DT) independently measured the ratio of positivity without knowledge of the clinical and pathological parameters or outcome of the patients. When >50% of tumor cells were stained, immunostaining was considered to be positive for KHDRBS3 in reference to the median value of the positivity. Inter‐observer differences were resolved by consensus review at a double‐headed microscope after independent reviews.