Immunohistochemical (IHC) labeling was performed at the IHC laboratory of the Department of Pathology. Briefly, 4-μm-thick tissue sections from TMAs and whole-section slides of cases showing MMR protein deficiency in TMAs were deparaffinized and hydrated in xylene and serially diluted alcohol solutions. Endogenous peroxidase was blocked by incubation in 3% H2O2 for 10 min; next, heat-induced antigen retrieval was performed. Primary antibodies with BenchMark auto-stainers (Ventana Medical Systems, Tucson, AZ, USA) were used following the manufacturer’s protocol. Primary antibodies for HER2 (4B5, mouse monoclonal, 1:8, Ventana), MLH1 (clone M1, mouse monoclonal, prediluted, Roche, Basel, Switzerland), MSH2 (clone G219-1129, mouse monoclonal, 1:1000, Cell Marque, Rocklin, CA, USA), MSH6 (clone 44, mouse monoclonal, 1:200, Cell Marque), and PMS2 (clone EP51, rabbit monoclonal, 1:100, Dako, Glostrup, Denmark) were used. After the evaluation of IHC-labeled TMA slides, additional immunolabeling was performed on sections on the whole-section slides if samples showed any loss of MMR proteins or an HER2 immunolabeling score of 2+ or 3+ on TMA slides.
Clone g219 1129
Manufactured by Cell Marque
Sourced in Switzerland, United Kingdom
Clone G219-1129 is a laboratory reagent used in immunohistochemistry applications. It functions as a monoclonal antibody that targets a specific protein or antigen.
Automatically generated - may contain errors
Lab products found in correlation
Antibody es05 clone, by Leica (2 mentions)
Benchmark auto stainers, by Roche (1 mentions)
Msh6 clone 44, by Cell Marque (1 mentions)
Clone m1, by Roche (1 mentions)
Prism 3130, by Thermo Fisher Scientific (1 mentions)
Genescan 2, by Thermo Fisher Scientific (1 mentions)
Ultraview universal dab detection kit, by Roche (1 mentions)
Mlh1 clone m1, by Roche (1 mentions)
Msi analysis system version 1, by Promega (1 mentions)
4 protocols using clone g219 1129
1
Immunohistochemical Evaluation of MMR and HER2
2
Microsatellite Instability and EBV Analysis
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MSI status of tumor tissue was determined in formalin-fixed paraffin-embedded tissue sections for MLH1 (antibody: ES05 clone; 1:100 dilution; Novocastra, UK) and MSH2 (clone G219-1129; 1: 500 dilution; CELL Marque; Rocklin, CA, USA) by IHC and PCR analysis of five markers with mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, and NR-27), as previously described. Briefly, each sense primer was end-labeled with FAM, HEX, or NED. Pentaplex PCR was performed, and the PCR products were run on an Applied Biosystems PRISM 3130 automated genetic analyzer. Allele sizes were estimated using GeneScan 2.1 software (Applied Biosystems, Foster City, CA, USA). Samples with allelic size variations in more than two microsatellites were considered MSI-H. EBV status was determined by EBER in situ hybridization using standard protocols [12 (link)].
3
Immunohistochemistry for Microsatellite Instability
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Immunohistochemistry staining and assessment for MLH1 (antibody: ES05 clone; 1:100 dilution; Novocastra) and MSH2 (clone G219-1129; 1:500 dilution; Cell Marque) in formalin-fixed paraffin-embedded tissue sections were used for determining the MSI status, as previously described (6 (link)). The loss of MLH1 and/or MSH2 expression defines the MSI-H status. EBV was evaluated using in-situ hybridization for EBV-encoded small RNA (17 (link)). Based on these results, we categorized patients into 4 groups: MSI-H, EBV positive, MSS/EBV negative, and unknown.
4
Comprehensive Microsatellite Instability Analysis
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Microsatellite instability (MSI) testing was performed in 14 cases using a fluorescent PCR-based assay (MSI Analysis system, Version 1.2, Promega, Madison, WI). Briefly, this test assessed 5 mononucleotide repeats (BAT25, BAT26, NR-21, NR-24, and MONO-27) and 2 pentanucleotide repeats (PentaC and PentaD) on genomic DNA and matched normal, where available. The fluorescently labeled PCR products were analyzed by capillary gel electrophoresis. MSI was determined if the tumor alleles showed a size difference ≥3 bp. Tumors with 2 or more microsatellite unstable markers were classified as MSI-H; the remainder were classified as microsatellite stable (MSS). Immunohistochemistry was performed for MLH1 (clone M1, predilute, Ventana), MSH6 (clone 44, predilute, Ventana), MSH2 (clone G219-1129, predilute, Cell Marque) and PMS2 (clone EPR3947, predilute, Cell Marque) using an autostainer (Benchmark UltraTM Ventana, Tuscon, AZ) after heat induced antigen retrieval (EDTA, pH 7.3). The ultraViewTM Universal DAB Detection kit (Ventana) was used for visualization. Absence of nuclear staining was evaluated in lesional tissue; adjacent stromal cells were used as an internal control.
Methods for DNA extraction, immunohistochemistry, flow cytometry, and Sanger sequencing are described in thesupplemental methods .
Methods for DNA extraction, immunohistochemistry, flow cytometry, and Sanger sequencing are described in the
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