Clone epr3947

Immunohistochemical staining against MMR proteins is a useful screening method in research materials with paraffin-embedded TMA-samples. In contrast to PCR-based methods, it also readily provides information on the inactivated gene. Immunohistochemical stainings (IHC) were performed using standard procedures. Shortly, 3,5 μm sections were cut from the TMA blocks. They were stained with monoclonal antibodies against MLH1 (Clone G168-15BD Pharmingen, dilution: 1:5), MSH2 (Clone G219-1129, BD Pharmingen, dilution: 1:200) and MSH6 (Clone EP49, Epitomoc, dilution: 1:200). The signal was detected with ultraVIEW Universal DAB Detection Kit. For PMS2, Clone EPR3947 (Ventana/Roche, ready to use antibody) was used and the signal was detected with OptiView Universal DAB Detection kit and amplification kit. To detect BRAF V600E mutation, BRAF RTU antibody (Clone VE1, Roche/Ventana) was used and the signal was detected with OptiView Universal DAB Detection kit. For ezrin staining, immunoglobulin G antibody to human ezrin (clone 3C12) [31 (link)] was used. All the stainings were performed with BenchMark XT (Ventana/Roche) using ultraVIEW Universal DAB Detection Kit (Ventana/Roche), except ezrin, which was done with LabVision immunoautomate (Thermo Fisher Scientific) using the Power Vision Plus poly HRP anti-mouse/rabbit/rat IgG detection kit.

One representative FFPE block of the cancer region in each case was chosen for IHC and ISH analysis. Unstained 4-um thick tissue sections were tested by IHC antibodies to MLH1 (Clone M1, ready-to-use; Roche), PMS2 (Clone EPR3947, ready-to-use; Roche), MSH2 (Clone 219-1129, ready-to-use; Roche) and MSH6 (Clone 44, ready-to-use; Roche) for detection of MMR status, as well as chromogenic ISH with EBV-encoded RNA (EBER, Ventana) probe to prove EBV infection, using Benchmark automated staining device (Ventana Medical Systems, Roche, Switzerland) according to the manufacturer' instructions. All IHC and ISH stained sections were reviewed and scored independently by two professional digestive pathologists (WLF and WRF) without knowledge of previous clinical or pathological parameters.
The slides were evaluated as follows: at least one of the MMR proteins (MLH1, MSH2, MSH6, and PMS2) with complete loss of nuclear reactivity in tumor cells but consistently preserved nuclear staining in background non-tumor cells was taken as d-MMR(aberrant expression). When the tumor cells demonstrated intact nuclear immunostaining of all four MMR proteins, the tumor was judged as p-MMR (normal expression). For EBER, tumors with strong blue-black nuclear staining were considered positive.