Clinical samples for IHC were obtained from CRC patients who received treatment at Gifu University (Gifu, Japan) from January 2010 to January 2016. Seven sets of naive primary tumors and remaining liver metastatic tumors after chemotherapy regimens, such as folinic acid, fluorouracil, and oxaliplatin (FOLFOX) containing anti-EGFR therapy, were used for this analysis 2 (link),3 (link). Informed consent was obtained from each patient.
IHC for GPNMB was performed according to standard procedures using the same antibody described here and a biotin-conjugated donkey anti-goat secondary antibody (Jackson Laboratories, West Grove, PA, USA) 19 (link). Goat IgG, a polyclonal isotype control (Abcam), was used as a negative control. For preliminary experiments, we created cell blocks containing three cell lines (GPNMB expression levels: high, SK-BR-3; moderate, MKN1; and low, LS174T). We used PROTEASE 1 (Ventana, Tuscon, AZ, USA) for antigen retrieval and determined the best conditions for GPNMB IHC (Fig.S1 ). Changes in GPNMB-staining intensity between each primary tumor and metastasis were determined by an experienced pathologist. This study was approved by the Central Ethics Committee of Gifu University.
IHC for GPNMB was performed according to standard procedures using the same antibody described here and a biotin-conjugated donkey anti-goat secondary antibody (Jackson Laboratories, West Grove, PA, USA) 19 (link). Goat IgG, a polyclonal isotype control (Abcam), was used as a negative control. For preliminary experiments, we created cell blocks containing three cell lines (GPNMB expression levels: high, SK-BR-3; moderate, MKN1; and low, LS174T). We used PROTEASE 1 (Ventana, Tuscon, AZ, USA) for antigen retrieval and determined the best conditions for GPNMB IHC (Fig.
