Pms2 clone m0r4g

FFPE blocks were obtained from the Pathology Department of Tokyo Metropolitan Bokutoh Hospital. Immunohistochemistry was performed using the Ventana BenchMark automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) with labeled streptavidin–biotin and visualized with 3,3′-diaminobenzidine. The primary antibodies used were anti-CD3 (clone LN10, Leica), -CD4 (clone SP35, Ventana), -CD8 (clone 4B11, Leica), -CD45RO (clone UCHL-1, Ventana), -FOXP3 (clone 236A/E7, Abcam), -CD20 (clone L26, Leica), -NKp46 (clone #195314, R&D), -CD68 (clone Kp-1, Dako), -CD163 (clone 10D6, Leica), -CD204 (clone SRA-E5, Transgenic), -Ki-67 (clone MIB-1, Dako), -PD-L1 (clone E1L3N, Cell Signaling), -MLH1 (clone ES05, Leica), -MSH2 (clone FE11, Dako), -MSH6 (clone Polyclonal (Rabbit), GeneTex) and -PMS2 (clone M0R4G, Leica). EBV-encoded small RNA in situ hybridization (EBER-ISH) was performed on paraffin sections using a fluorescein isothiocyanate (FITC)-labeled peptide nucleic acid probe (Y5200; Dako, Glostrup, Denmark) and anti-FITC antibody (V0403, Dako). Slides were digitized with a Nanozoomer 2.0-HT virtual slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) and observed in the NDP.view2 software (Hamamatsu Photonics). The density of immune cells was analyzed by Tissue Studio 2.0 software (Definiens, Munich, Germany).

Immunohistochemistry (IHC) for the four MMR proteins (MLH1, PMS2, MSH2 and MSH6) was performed on FFPE slides from more recent specimens, including 33 AWMC cases and 88 AC cases. The antibodies used were: MLH1 (clone ES05, Leica), MSH2 (clone 25D12, Leica), MSH6 (clone PU29, Leica) and PMS2 (clone M0R4G, Leica). Normal colon epithelium and infiltrating lymphocytes were used as internal positive controls. dMMR was defined as complete nuclear loss of expression of one or more of these proteins.