Clone g219 1129

Immunohistochemistry (IHC) staining of non-tumor- and tumor-FFPE samples for MMR proteins MLH1, MSH2, MSH6, and PMS2 was performed according to established laboratory protocol at the Department of Pathology at the Institute of Oncology Ljubljana, Slovenia. Briefly, IHC staining was performed on 2 to 4 µm FFPE tissue sections using fully automated IHC system Ventana Benchmark XT (Ventana Roche, Oro Valley, USA). For this purpose, commercially available monoclonal antibodies for MLH1 (clone ES05, #M3640) (DAKO Agilent, Santa Clara, USA), PMS2 (clone EP51, #M3647) (DAKO Agilent), MSH2 (clone G219-1129, #556349) (BD Pharmingen, Franklin Lakes, USA) and MSH6 (clone SP93, #287R) (CellMarque, Rocklin, USA) were used. Primary antibodies were visualized using a three–step multimer detection system OptiView DAB IHC Detection Kit (Ventana Roche) following the manufacturer's protocol. MLH1 and PMS2 detection was enhanced using OptiVew Amplification Kit (Ventana Roche). All protocols passed external quality assessment in NEQAS and NORDIQC. Normal appendix and colon carcinomas with confirmed protein expression or loss of protein expression were used as controls.

Immunohistochemical staining of DNA mismatch repair proteins as well as of proteins CTNNB1, CCND1 (cyclin D1) and c-myc in tumor tissue was performed as follows: 3–4 μm slides were cut and stained for MLH1 (Monoclonal Mouse Anti-Human; Clone ES05, dilution 1 : 10, Dako), MSH2 (Monoclonal Mouse Anti-Human; Clone G219-1129, dilution 1 : 200, BD Biosciences), MSH6 (Monoclonal Rabbit Anti-Human; Clone EP49, dilution 1 : 1000 Dako), PMS2 (Monoclonal Mouse Anti-Human; Clone A 16–4, dilution 1 : 100, BD Biosciences) to assess the reactivity in the nuclei of tumor cells as well as for CTNNB1 (Monoclonal Mouse Anti-Human; clone β-Catenin-1, ready to use, Dako), CCND1 (Monoclonal Rabbit Anti-Human; Clone EP12, ready to use, Dako) and c-myc (Monoclonal Rabbit Anti-Human; Clone Y69, 1:100 dilution, Abcam). Immunostains were developed according to an antigen retrieval treatment using a detection system suitable for the Dako Autostainer Link 48 (EnVision FLEX, Dako).