Msh6 clone ep49

Manufactured by Agilent Technologies

Sourced in Denmark

MSH6 (clone EP49) is a monoclonal antibody used for the detection of the MSH6 protein in biological samples. MSH6 is a component of the DNA mismatch repair system and plays a role in recognizing and repairing DNA mismatches.

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Lab products found in correlation

Pms2 clone ep51, by Agilent Technologies (7 mentions) Msh2 clone fe11, by Agilent Technologies (6 mentions) Mlh1 clone es05, by Agilent Technologies (5 mentions) P53 clone do 7, by Agilent Technologies (3 mentions) Bond polymer refine detection kit, by Leica (3 mentions) Bond max system, by Leica (2 mentions) Envision kit, by Agilent Technologies (2 mentions) E cadherin clone nch38, by Agilent Technologies (1 mentions) Bond max, by Leica (1 mentions) Cd8 clone c8 144b, by Agilent Technologies (1 mentions) Pd l1 clone 22c3, by Agilent Technologies (1 mentions) Bond max automated ihc stainer, by Leica (1 mentions) Bond 3 stainer, by Leica (1 mentions) Iprep chargeswitch forensic kit, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Clone EP49 (8 citations) MSH6 (clone EP49 M3646) (3 citations)

8 protocols using msh6 clone ep49

Immunohistochemical Evaluation 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: PMS2 (clone EP51; dilution: 1:50 Dako), MSH6 (clone EP49; dilution: 1:50; Dako), E-cadherin (clone NCH-38; dilution: 1:50; Dako), and p53 (clone DO-7; dilution: 1:150; Dako). IHC slides were jointly evaluated by three pathologists (VA, GP, and MF).
Mismatch Repair defective status was assessed by testing PMS2 and MSH6, and samples were defined as dMMR when one or both proteins resulted negative [15 (link)]. In case of protein loss, the dominant component of the heterodimer (i.e., MLH1 for PMS2 and MSH2 for MSH6; Dako) was tested.
p53 was considered as aberrant in the presence of complete loss or diffuse and strong nuclear immunostaining in dysplastic cells [9 (link), 16 (link)].
E-cadherin expression was considered altered in the presence of complete loss or markedly reduced membranous staining (> 30%), regardless of nuclear/cytoplasmic staining [9 (link)].

Angerilli V., Pennelli G., Galuppini F., Realdon S., Fantin A., Savarino E., Farinati F., Mastracci L., Luchini C, & Fassan M. (2022). Molecular subtyping of gastroesophageal dysplasia heterogeneity according to TCGA/ACRG classes. Virchows Archiv, 481(4), 545-552.

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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 Evaluation of MMR Proteins

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IHC was carried out on 2 um thick TMA sections with the following antibodies and conditions: MLH1, clone ES05 (Monosan, Uden, Netherlands) at 1:80 dilution; MSH2, clone 25D12 (Novocastra/Leica Microsystems, Wetzlar, Germany) at 1:40 dilution; MSH6, clone EP49 (DAKO, Glostrup, Denmark) at 1:60 dilution; and, PMS2, clone M0R4G (Novocastra/Leica Microsystems) at 1:50 dilution. All tests were performed using a Bond Max™ autostainer (Leica Microsystems) with diaminobenzidine as chromogen for protein-antibody complex visualization. Stains were evaluated by two pathologists (G.R. and E.V.) for all tumor and normal cores, along with external controls for assessing method performance. Each core was evaluated for nuclear staining intensity and distribution of positive cells at 200× and 400× [8 (link)]. Tumors were scored for (a) the incidence of positive cells as 0 (<10 % positive), 1 (10–30 % positive), 2 (30–70 % positive) and 3 (>70 % positive); and (b) for staining intensity as 0 (negative), 1 (mild), 2 (intermediate) and 3 (strong), in comparison to internal controls (lymphocytes, normal epithelia) [9 (link)–11 (link)]. Scores for each core were recorded. For the purposes of the present study, tumors were classified as positive for incidence and intensity categories 1–3 (≥10 % positive nuclei with mild to strong intensity).

Pectasides D., Karavasilis V., Papaxoinis G., Gourgioti G., Makatsoris T., Raptou G., Vrettou E., Sgouros J., Samantas E., Basdanis G., Papakostas P., Bafaloukos D., Kotoula V., Kalofonos H.P., Scopa C.D., Pentheroudakis G, & Fountzilas G. (2015). Randomized phase III clinical trial comparing the combination of capecitabine and oxaliplatin (CAPOX) with the combination of 5-fluorouracil, leucovorin and oxaliplatin (modified FOLFOX6) as adjuvant therapy in patients with operated high-risk stage II or stage III colorectal cancer. BMC Cancer, 15, 384.

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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 Analysis of Mismatch Repair Proteins in FFPE Tumors

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Formalin‐fixed paraffin‐embedded tumour blocks were used to create tissue microarrays (TMA) containing two to three 2‐mm cores of the tumour. Immunohistochemical staining on 4 μm TMA sections was performed on the automated BOND‐III stainer (Leica Biosystems, Amsterdam, the Netherlands) with alkaline antigen retrieval (solution ER2; Leica Biosystems) for the four mismatch repair proteins MLH1 (clone S05; 1:60; Leica Biosystems), PMS2 (clone EP51; 1:30; Dako, Glostrup, Denmark), MSH2 (clone FE11; 1:15; Dako) and MSH6 (clone EP49; 1:50; Dako). dMMR, defined as unequivocal absence of one or more of the MMR stains in the cell nuclei with a positive internal control present in the same tissue section, is considered indicative of MSI. Tumours with positive nuclear staining of all four proteins were pMMR and were considered MSS.

van Zwam P.H., Vink‐Börger E.M., Bronkhorst C.M., de Bruine A.P., van der Wurff A.A., Rutten H.J., Lemmens V.E., Nagtegaal I.D, & Hugen N. (2022). Prognosis of mucinous colon cancer is determined by histological biomarkers rather than microsatellite instability. Histopathology, 82(2), 314-323.

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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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