Immunohistochemical analyses were performed on 3 μm thick sections in all tumours classified as microsatellite-unstable by polymerase chain reaction-based methods. Briefly, the slides were pretreated by boiling for 10 min with a microwave in target retrieval buffer (pH 9, Dako, Hamburg, Germany) before application of monoclonal antibodies specific for MSH2 (clone FE11, dilution 1:100, Calbiochem, Darmstadt, Germany), MLH1 (clone G168-15, dilution 1:100, BD Biosciences, San Diego, CA, USA), MSH6 (1:200; clone 44; BD Biosciences, San Diego, CA, USA), and PMS2 (ready to use; clone EPR3947, Cell Marque, Sigma Aldrich, St. Louis, Missouri, USA). An immunoperoxidase method was used to visualise bound antibodies with 3-amino-9-ethylcarbazole (Dako) as chromogen.
Mlh1 clone g168 15
Manufactured by BD
Sourced in Germany, United States
MLH1 (clone G168-15) is an antibody used in laboratory research. It binds to the MLH1 protein, which is involved in DNA mismatch repair processes. The core function of this antibody is to detect and identify the presence of the MLH1 protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
3 amino 9 ethylcarbazole, by Agilent Technologies (1 mentions)
Msh6 clone 44, by BD (1 mentions)
Clone ep49, by Novus Biologicals (1 mentions)
Clone a16 4, by BD (1 mentions)
Target retrieval buffer, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
5 protocols using mlh1 clone g168 15
1
Immunohistochemical Analysis of MSI Tumors
2
Evaluating DNA Mismatch Repair Proteins
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DNA MMR protein expression (MLH1, MSH2, and MSH6) was evaluated by IHC. IHC was performed on paraffin sections of normal and tumor tissues (4 µm thick) using mouse monoclonal antibodies specific for each MMR protein as follows: MLH1 (clone G168-15, 1:200; BD Pharmingen, San Diego, CA, USA), MSH2 (clone FE11, 1:400; Calbiochem, La Jolla, CA, USA), and MSH6 (clone 44, 1:400; BD Transduction Laboratories, San Diego, CA, USA). MMR protein expression was described as negative for absent or <10% nuclear staining, and positive for ≥10% nuclear staining. Normal colonic epithelium adjacent to the tumor and lymphocytes served as positive controls.
3
Immunohistochemistry for MMR and PD-L1
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Immunohistochemistry was done on 2-µm paraffin sections. Briefly, the slides were pretreated by boiling for 15 min in a microwave oven in citrate buffer (pH 6). First antibodies specific for MMR proteins and PD-L1 were as follows: MSH2 (clone FE11, dilution 1:100; Calbiochem), MLH1 (clone G168-15, dilution 1:100; BD Pharmingen), MSH6 (1:100; clone EP49; Novus Biologicals), PMS2 (1.50; clone A16-4; BD Pharmingen) and PD-L1 (clone SP263; Ventana Benchmark Ultra, Tuscon). Visualization was carried out following a standard avidin–biotin method (ABC; Vectastain).
4
Immunohistochemical Analysis of MSI Markers
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Immunohistochemical analyses were performed on 3 μm thick sections in all tumors classified as MSI by PCR-based methods. Briefly, the slides were pretreated by boiling for 10 min with a microwave in target retrieval buffer (pH 9, Dako, Hamburg, Germany) before application of monoclonal antibodies specific for MSH2 (clone FE11, dilution 1:100, Calbiochem, Darmstadt, Germany), MLH1 (clone G168–15, 1:100, BD Biosciences, San Diego, CA, USA), MSH6 (clone 44; 1:200; BD Biosciences, San Diego, CA, USA), and PMS2 (clone EPR3947, ready to use; Cell Marque, Sigma Aldrich, St. Louis, Missouri, USA). An immunoperoxidase method was used to visualize bound antibodies with DAB (3-amino-9-ethylcarbazole, Dako) as chromogen.
5
Microsatellite Instability Immunohistochemistry
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Immunohistochemical staining of MLH1 (clone G168‐15; BD Biosciences, San Jose, California, USA) and MSH2 (FE11; Invitrogen, Carlsbad, California, USA) were used to verify retrospectively the microsatellite instability status, as reported previously14 . Tumour cells were judged to be negative for protein expression only when they lacked staining in a sample in which healthy colonocytes and stroma cells were stained. The normal colonic crypt epithelium adjoining the tumour was used as an internal control. Both MLH1 and MSH2 proteins should stain positively in nuclei when they are expressed15 . Cancers with negative MLH1 or MSH2 expression were considered to have DNA mismatch repair deficiency.
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