Clone g168 15

PD-L1 expression was assessed by IHC staining, using SP263 (Ventana Benchmark Ultra, Tuscon, AZ, USA), the 22C3 pharmDx kit (Agilent Technologies, Santa Clara, CA, USA), or E1L3N XP Rabbit mAb (Cell Signaling Technology, Danvers, MA, USA). If more than 1% of viable tumor cells had PD-L1, they were considered PD-L1-positive [18 (link)]. The tumor proportion score (TPS) was defined as the percentage of viable tumor cells showing partial or complete membrane-staining at any intensity [19 (link)]. The combined positive score (CPS) was defined as the number of PD-L1-positive cells (tumor cells, lymphocytes, and macrophages), divided by the total number of viable tumor cells, and multiplied by 100 [20 (link)].
The microsatellite stability was assessed using antibodies specific for mismatch repair proteins, including mutL homolog 1 (1:10; clone G168-15; BD Pharmingen, San Jose, CA, USA), mutS homolog (MSH)-2 (1:100; clone FE11; Calbiochem, San Diego, CA, USA), MSH6 (1:100; clone EP49; Novus Biologicals, Centennial, CO, USA), and PMS1 homolog 2 (1:50; clone A16-4; BD Pharmingen).

We examined MMR-protein expression for the MLH1, MSH2, MSH6, and PMS2 proteins in primary tumors from 317 CRC and 138 EC patients by IHC. Thin (5 µm) sections of representative blocks were deparaffinized and dehydrated using an ethanol gradient. Following antigen retrieval in citrate buffer (pH 6.0), endogenous peroxidase was blocked with 3% H₂O₂. Thereafter, slides were incubated overnight in the presence of purified mouse monoclonal antibodies against MLH1 (clone G168-15; 1:50; BD Pharmingen, San Diego, CA, USA), MSH2 (clone G219-1129; 1:200; BD Pharmingen), MSH6 (clone 44/MSH6; 1:100; BD Pharmingen), and PMS2 (clone A16-4; 1:200; BD Pharmingen). Additional incubations were performed with a biotin-conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA), the avidin–biotin–peroxidase complex (Vector Laboratories, Burlingame, CA, USA), and with biotinyl tyramide, followed by streptavidin peroxidase. Diaminobenzidine was used as a chromogen, and hematoxylin was used as a nuclear counterstain. Tumor cells were scored as negative for MMR-protein expression only if the epithelial cells within the tumor tissue lacked nuclear staining while the surrounding stromal cells were positive for MMR staining. Tumor tissue with all MMR proteins present were defined as pMMR, and those showing deficiency in at least one of the four MMR proteins were defined as dMMR.