Msh2 clone g219 1129

IHC was done on all CRC and EC tissue samples to detect the loss of MMR protein expression as a golden standard test using the following antibodies: MLH1 (clone ES05, diluted 1:50; Dako/Agilent, Santa Clara, CA), MSH2 (clone G219-1129, diluted 1:400; BD Biosciences, San Jose, CA), MSH6 (clone EPR3945, diluted 1:200; Abgent, San Diego, CA), and PMS2 (clone EP51, diluted 1:50; Dako/Agilent, Santa Clara). The MSH2 and MSH6 stainings were performed with Ventana BenchMark ULTRA immunostainer (Roche, Ventana Medical Systems, Tucson, AZ, USA) utilizing OptiView DAB kit (760-700, Ventana/Roche). MLH1 and PMS2 stainings were performed with Autostainer (Agilent/Dako, Santa Clara, USA) utilizing BrightVision detection kit (DPVB110HRP, Immunologic, WellMed, Duiven, the Netherlands). The loss of one or more MMR protein was defined as a dMMR, and the expression of all four MMR proteins was defined as proficient MMR (pMMR). Negative MMR protein expression was considered valid if nuclear staining in the tumor cells was absent with positive external (normal colon mucosa) and internal control staining (stromal nonneoplastic cells).

IHC was performed by standard procedures [46 (link)] with the following primary antibodies: MLH1 (clone ES05; 75 mg/l; Dako North America, Inc. CA), MSH2 (clone G219-1129; 0.5 mg/ml; BD Pharmingen), MSH6 (clone EP49, AC00-47, Epitomics, Burlingame, CA), and PMS2 (clone EPR3947, 0.324 mg/ml, Abcam, Cambridge, UK). Negative cancer cell immunostaining was interpreted to indicate inactivation of the respective MMR gene.