Immunohistochemical Analysis of MMR Proteins

We examined protein expression for MLH1, MSH2, PMS2, and MSH6 in 138 tumor tissues by immunohistochemical (IHC) staining using DAKO EnVision System-HRP polymer system kit (DakoCytomation California, Inc., Carpinteria, CA, USA). Staining was performed manually with FFPE specimens. Thin (5 μm) sections of representative blocks were deparaffinized and dehydrated using gradient solvents. Following antigen retrieval in the 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, BD Pharmingen, San Diego, CA, USA; dilution 1:50), MSH2 (clone G219-1129, BD Pharmingen; dilution 1:200), PMS2 (clone A16-4, BD Pharmingen; dilution 1:200), and MSH6 protein (clone 44/MSH6, BD Pharmingen; dilution 1:100), respectively. A further incubation was performed with a secondary antibody and the avidin–biotin–peroxidase complex (Vector Laboratories, Burlingame, CA, USA) and then incubated with biotinyltyramide, followed by streptavidin–peroxidase. Diaminobenzidine was used as a chromogen and hematoxylin as a nuclear counterstain. Tumor cells were scored negative for MMR protein expression only if the epithelial cells within the tumor tissue lacked nuclear staining, while the surrounding stromal cells still showed positive staining. Samples showing proficiency in expression of all MMR proteins were defined as pMMR, and samples showing deficiency in at least one of the four MMR proteins were defined as dMMR.