Mlh1 clone es05

Manufactured by Agilent Technologies

Sourced in Denmark

MLH1 (clone ES05) is a lab equipment product offered by Agilent Technologies. It is a primary antibody that can be used for the detection of the MLH1 protein in immunohistochemical (IHC) applications.

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Clone ES05 (7 citations)

9 protocols using mlh1 clone es05

Immunohistochemical Analysis of MMR Proteins

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IHC was done on all CRC and EC tissue samples to detect the loss of MMR protein expression as a golden standard test using the following antibodies: MLH1 (clone ES05, diluted 1:50; Dako/Agilent, Santa Clara, CA), MSH2 (clone G219-1129, diluted 1:400; BD Biosciences, San Jose, CA), MSH6 (clone EPR3945, diluted 1:200; Abgent, San Diego, CA), and PMS2 (clone EP51, diluted 1:50; Dako/Agilent, Santa Clara). The MSH2 and MSH6 stainings were performed with Ventana BenchMark ULTRA immunostainer (Roche, Ventana Medical Systems, Tucson, AZ, USA) utilizing OptiView DAB kit (760-700, Ventana/Roche). MLH1 and PMS2 stainings were performed with Autostainer (Agilent/Dako, Santa Clara, USA) utilizing BrightVision detection kit (DPVB110HRP, Immunologic, WellMed, Duiven, the Netherlands). The loss of one or more MMR protein was defined as a dMMR, and the expression of all four MMR proteins was defined as proficient MMR (pMMR). Negative MMR protein expression was considered valid if nuclear staining in the tumor cells was absent with positive external (normal colon mucosa) and internal control staining (stromal nonneoplastic cells).

Ukkola I., Nummela P., Pasanen A., Kero M., Lepistö A., Kytölä S., Bützow R, & Ristimäki A. (2021). Detection of microsatellite instability with Idylla MSI assay in colorectal and endometrial cancer. Virchows Archiv, 479(3), 471-479.

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Immunohistochemical Analysis of CRC Samples

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Formalin fixed, paraffin-embedded samples of CRC or adenomas were selected for immunohistochemical studies. Immunohistochemical evaluation, on 3 μm thick sections, was done using the following Ready-to-use antibodies MLH1 (clone ES05), PMS2 (clone EP51, MSH2 (clone FE11) and MSH6 (EP49) from Agilent following the manufacturer’s instructions. The staining took place on the Omnis from Agilent utilizing the EnVision Flex + detection kit (GV800). The sections were counterstained with hematoxylin.
Some samples have been analyzed in other departments of pathology, and historically only MLH1 and MSH2 or MLH1, MSH2, and MSH6 were analyzed. These samples might have been analyzed using other kits, however, all samples have been analyzed as part of a clinical evaluation by experienced pathologists.

Djursby M., Madsen M.B., Frederiksen J.H., Berchtold L.A., Therkildsen C., Willemoe G.L., Hasselby J.P., Wikman F., Okkels H., Skytte A.B., Nilbert M., Wadt K., Gerdes A.M, & van Overeem Hansen T. (2020). New Pathogenic Germline Variants in Very Early Onset and Familial Colorectal Cancer Patients. Frontiers in Genetics, 11, 566266.

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Immunohistochemical Analysis of DNA Mismatch Repair Proteins

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Representative non-necrotic central areas of each tumour were marked on H&E slides. Two representative cores of 1.2 mm of diameter were taken from the selected areas of the paraffin block. The tissue cores were arrayed into a receptor paraffin block using a tissue microarray (TMA) workstation (Beecher Instruments, Silver Spring, MD, USA) as described previously [36 (link)].
Immunohistochemistry was performed on 4 μm sections of the TMA blocks using the following primary antibodies: MLH1 (clone ES05, Dako, Glostrup, Denmark; prediluted), PMS2 (clone EP51, Dako, prediluted), MSH2 (clone FE11, Dako, prediluted), MSH6 (clone EP49, Dako, prediluted). Staining was performed using an OMNIS autostainer (Dako) according to the manufacturer´s instructions.
Analysis of immunohistochemical stains was performed as described previously [36 (link)]. Whole slide immunohistochemistry was performed in doubtful cases.

Ruz-Caracuel I., Ramón-Patino J.L., López-Janeiro Á., Yébenes L., Berjón A., Hernández A., Gallego A., Heredia-Soto V., Mendiola M., Redondo A., Peláez-García A, & Hardisson D. (2019). Myoinvasive Pattern as a Prognostic Marker in Low-Grade, Early-Stage Endometrioid Endometrial Carcinoma. Cancers, 11(12), 1845.

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Immunohistochemical Assessment of MMR Proteins

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Following standard procedures, MMR-IHC was performed to assess the expression of MMR proteins (MLH1, MSH2, MSH6, and PMS2) in tumors of all EC patients. An appropriate paraffin-embedded tissue was cut to 4 μm-thickness. The tissue sections were deparaffinized with xylene and rehydrated in graded alcohol. Antigen retrieval was performed in 10 mmol/L Tris-EDTA buffer (pH 9.0) in a microwave oven for 20 minutes. The sections were cooled to room temperature. The primary antibody was added overnight at 4°C. The following primary antibodies were used: MLH1 (clone ES05; dilution 1:50; Dako, Glostrup, Denmark), MSH2 (clone FE11; dilution 1:50; Dako), MSH6 (clone EP49; dilution 1:50; Dako), and PMS2 (clone EP51; dilution 1:40; Dako). The antigen-antibody reaction was visualized using the Envision kit (Dako). The slides were counterstained with hematoxylin. Adjacent normal endometrium and lymphocytes in the section were used as internal positive controls. Representative IHC photos of MMR expression were shown in Fig. 1B. According to the standard screening methods for LS, cases with a complete absence of nuclear staining in whole sections were judged as “loss of MMR protein expression.”

Kaneko E., Sato N., Sugawara T., Noto A., Takahashi K., Makino K, & Terada Y. (2021). MLH1 promoter hypermethylation predicts poorer prognosis in mismatch repair deficiency endometrial carcinomas. Journal of Gynecologic Oncology, 32(6), e79.

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Immunohistochemical Profiling of Colorectal Cancer

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IHC was performed using the bond polymer refine detection kit (Leica Biosystems, Newcastle upon Tyne, UK) in the BOND-MAX system (Leica Biosystems). Four-micrometer-thick FFPE sections were incubated with the following primary antibodies: MLH1 (clone ES05; Dako), PMS2 (clone EP51; Dako), MSH2 (clone FE11; Dako), MSH6 (clone EP49; Dako), ^V600EBRAF (clone VE1; Ventana), and p53 (clone DO-7; Dako).
p53 and ^V600EBRAF immunostaining were performed only if a TP53 or BRAF mutation was found by mutational analysis.
Nuclear immunostaining for MLH1, PMS2, MSH2, and MSH6 was evaluated following the GIPAD-SIAPeC criteria [6 (link)] to identify mismatch repair deficiency (MMRd) and mismatch repair proficiency (MMRp) profile.
p53 was considered as aberrant in the presence of complete loss or diffuse and strong nuclear immunostaining in neoplastic cells.
^V600EBRAF was considered positive in the presence of cytoplasmic positivity.

Angerilli V., Parente P., Businello G., Vanoli A., Paudice M., Perrone G., Munari G., Govoni I., Neri G., Rebellato E., Parrella P., Grillo F., Mastracci L, & Fassan M. (2023). Colorectal adenosquamous carcinoma: genomic profiling of a rare histotype of colorectal cancer. Virchows Archiv, 482(5), 879-885.

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Immunohistochemical Assessment of MMR Proteins

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MMR-IHC was performed on tumors of all 360 EC patients to assess MMR protein (MLH1, MSH2, MSH6, and PMS2) expression, according to standard procedure. An appropriate paraffin-embedded tissue was cut at 4 μm thickness. The tissue sections were deparaffinized in xylene and rehydrated in graded alcohol. Subsequently, antigen retrieval was performed in 10 mmol/L Tris-EDTA buffer (pH 9.0) in a microwave oven for 20 minutes. These sections were allowed to cool at room temperature. Thereafter, the primary antibodies were applied overnight at 4°C. The primary antibodies were MLH1 (clone ES05, dilution 1:50; Dako), MSH2 (clone FE11, dilution 1:50; Dako), MSH6 (clone EP49, dilution 1:50; Dako), and PMS2 (clone EP51, dilution 1:40; Dako). The antigen-antibody reaction was visualized with the Envision kit (Dako). The slides were counterstained with hematoxylin. Adjacent normal endometrium and lymphocytes in the slides were used as an internal positive control. We judged the complete absence of nuclear staining in the tumor cells as loss of MMR protein expression.

Kato A., Sato N., Sugawara T., Takahashi K., Kito M., Makino K., Sato T., Shimizu D., Shirasawa H., Miura H., Sato W., Kumazawa Y., Sato A., Kumagai J, & Terada Y. (2016). Isolated Loss of PMS2 Immunohistochemical Expression is Frequently Caused by Heterogenous MLH1 Promoter Hypermethylation in Lynch Syndrome Screening for Endometrial Cancer Patients. The American Journal of Surgical Pathology, 40(6), 770-776.

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Immunohistochemical Staining Protocol for Biomarkers

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The Bond Polymer Refine Detection Kit (Leica Biosystems) on BOND‐MAX automated IHC stainer (Leica Biosystems) was used for immunohistochemical stainings. The following primary antibodies were used according to the manufacturer's directions: MLH1 (clone ES05, 1:100; Dako), MSH2 (clone FE11, 1:100; Dako), MSH6 (clone EP49, 1:100; Dako), PMS2 (clone EP51, 1:100; Dako), PD‐L1 (clone 22C3, 1:50; Dako), PD‐L2 (clone 176611, 1:1000; R&D Systems, Inc.), p53 (clone DO7, 1:50; Dako), CD80 (clone 37711, 1:40; R&D Systems, Inc.), CD8 (clone C8/144B, 1:200; Dako), LAMP1 (clone H5G11, 1:50; Santa Cruz Biotechnologies) and HER2 (Hercept test; Dako).

Kotsafti A., Fassan M., Cavallin F., Angerilli V., Saadeh L., Cagol M., Alfieri R., Pilati P., Castoro C., Castagliuolo I., Scarpa M, & Scarpa M. (2022). Tumor immune microenvironment in therapy‐naive esophageal adenocarcinoma could predict the nodal status. Cancer Medicine, 12(5), 5526-5535.

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Immunohistochemical Assessment of DNA Mismatch Repair

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IHC was performed by standard procedures [46 (link)] with the following primary antibodies: MLH1 (clone ES05; 75 mg/l; Dako North America, Inc. CA), MSH2 (clone G219-1129; 0.5 mg/ml; BD Pharmingen), MSH6 (clone EP49, AC00-47, Epitomics, Burlingame, CA), and PMS2 (clone EPR3947, 0.324 mg/ml, Abcam, Cambridge, UK). Negative cancer cell immunostaining was interpreted to indicate inactivation of the respective MMR gene.

Valo S., Kaur S., Ristimäki A., Renkonen-Sinisalo L., Järvinen H., Mecklin J.P., Nyström M, & Peltomäki P. (2015). DNA hypermethylation appears early and shows increased frequency with dysplasia in Lynch syndrome-associated colorectal adenomas and carcinomas. Clinical Epigenetics, 7(1), 71.

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Colorectal Cancer Microsatellite Instability Assay

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Microsatellite instability or mismatch repair statuses were performed as part of the diagnosis or for the purpose of the study by polymerase chain reaction (PCR) or immunohistochemistry, respectively (25 tumors were tested by immunohistochemistry only, the remaining by immunohistochemistry and PCR). The microsatellite instability status was determined using a pentaplex PCR with five markers: BAT-25, BAT-26, NR-21, NR-22, and NR-24 [18] . Briefly, genomic DNA was extracted from 10 µm thick tissue sections of formalin-fixed, paraffin-embedded colorectal tumor tissue after manual macrodissection using iPrepTM Char-geSwitch® Forensic kit (Invitrogen), and according to the manufacturer's instructions. A colorectal cancer was considered as microsatellite instable if at least 2 of these 5 markers showed microsatellite instability [19] . The mismatch repair status was assessed by immunohistochemistry using the following antibodies: MLH1 (clone ES05, Dako), MSH2 (clone FE11, Dako), MSH6 (clone EP49, Dako), and PMS2 (clone EP51, Dako).

Eugène J., Jouand N., Ducoin K., Dansette D., Oger R., Deleine C., Leveque E., Meurette G., Podevin J., Matysiak T., Bennouna J., Bezieau S., Volteau C., Thomas W.E.A., Chetritt J., Kerdraon O., Fourquier P., Thibaudeau E., Dumont F., Mosnier J.F., Toquet C., Jarry A., Gervois N, & Bossard C. (2020). The inhibitory receptor CD94/NKG2A on CD8⁺ tumor-infiltrating lymphocytes in colorectal cancer: a promising new druggable immune checkpoint in the context of HLAE/β2m overexpression. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 33(3).

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