Msh2 clone fe11

Manufactured by Agilent Technologies

Sourced in Denmark

MSH2 (clone FE11) is a laboratory reagent used for the detection of the MSH2 protein in various biological samples. The MSH2 protein is a key component of the DNA mismatch repair system, which plays a crucial role in maintaining genomic stability. This product can be used in research applications, such as immunohistochemistry and Western blotting, to study the expression and localization of the MSH2 protein.

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Lab products found in correlation

Pms2 clone ep51, by Agilent Technologies (7 mentions) Mlh1 clone es05, by Agilent Technologies (6 mentions) Msh6 clone ep49, by Agilent Technologies (6 mentions) Envision kit, by Agilent Technologies (2 mentions) P53 clone do 7, by Agilent Technologies (2 mentions) Bond polymer refine detection kit, by Leica Biosystems (2 mentions) Omnis autostainer, by Agilent Technologies (1 mentions) Tma workstation, by Beecher Instruments (1 mentions) Bond max system, by Leica Biosystems (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 Biosystems (1 mentions) Dab kit, by Agilent Technologies (1 mentions) Abc kit, by Vector Laboratories (1 mentions) Bond 3 stainer, by Leica Biosystems (1 mentions) Pd l1 clone e1l3n, by Cell Signaling Technology (1 mentions) Anti cd3 clone ln10, by Leica camera (1 mentions) Mlh1 clone es05, by Leica camera (1 mentions) Pms2 clone m0r4g, by Leica camera (1 mentions) Ventana benchmark, by Roche (1 mentions)

9 protocols using msh2 clone fe11

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 Analysis of MMR and TLR4 Proteins

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Histology sections (3 m), obtained from formalin fixed, paraffin embedded specimens, were stained with hematoxylin-eosin. Paraffin-embedded tumors (n = 89) were analyzed for MLH1, MSH2, MSH6, and PMS2 proteins (Lanza et al., 2006 (link)). Immunohistochemical analyses were performed using standard procedures. Primary antibodies used were for MLH1 (clone ES05; Dako, Glostrup, Denmark), PMS2 (clone EP51; Dako), MSH6 (clone EP49; Dako), MSH2 (clone FE11; Dako), TLR4 (clone 76B357.1; Abcam, Cambridge, United Kingdom). Immunocomplexes were detected using an avidin-biotin-peroxidase conjugate and 3-3 di-aminobenzidine tetrahydrochloride chromogen as a substrate (ABC Kit, Vector Laboratories, Burlingame, CA, United States; and DAB kit Dako, Glostrup, Denmark). Slides were scored by a GI expert pathologist (F.C.) as either positive or negative based on the presence or absence of nuclear staining for each MMR protein in the tumor cells. Each slide contained a unique number that enabled blinding with respect to patient identity and clinical characteristics. TLR4 expression was graded on a semi quantitative scale (negative, low, moderate or high). Ten random fields (x63) from each sample were examined.

Scarpa M., Ruffolo C., Kotsafti A., Canal F., Erroi F., Basato S., DallAgnese L., Fiorot A., Pozza A., Brun P., Bassi N., Dei Tos A., Castoro C., Castagliuolo I, & Scarpa M. (2021). MLH1 Deficiency Down-Regulates TLR4 Expression in Sporadic Colorectal Cancer. Frontiers in Molecular Biosciences, 8, 624873.

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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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Comprehensive Immunophenotyping of FFPE Samples

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FFPE blocks were obtained from the Pathology Department of Tokyo Metropolitan Bokutoh Hospital. Immunohistochemistry was performed using the Ventana BenchMark automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) with labeled streptavidin–biotin and visualized with 3,3′-diaminobenzidine. The primary antibodies used were anti-CD3 (clone LN10, Leica), -CD4 (clone SP35, Ventana), -CD8 (clone 4B11, Leica), -CD45RO (clone UCHL-1, Ventana), -FOXP3 (clone 236A/E7, Abcam), -CD20 (clone L26, Leica), -NKp46 (clone #195314, R&D), -CD68 (clone Kp-1, Dako), -CD163 (clone 10D6, Leica), -CD204 (clone SRA-E5, Transgenic), -Ki-67 (clone MIB-1, Dako), -PD-L1 (clone E1L3N, Cell Signaling), -MLH1 (clone ES05, Leica), -MSH2 (clone FE11, Dako), -MSH6 (clone Polyclonal (Rabbit), GeneTex) and -PMS2 (clone M0R4G, Leica). EBV-encoded small RNA in situ hybridization (EBER-ISH) was performed on paraffin sections using a fluorescein isothiocyanate (FITC)-labeled peptide nucleic acid probe (Y5200; Dako, Glostrup, Denmark) and anti-FITC antibody (V0403, Dako). Slides were digitized with a Nanozoomer 2.0-HT virtual slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) and observed in the NDP.view2 software (Hamamatsu Photonics). The density of immune cells was analyzed by Tissue Studio 2.0 software (Definiens, Munich, Germany).

Saito N., Sato Y., Abe H., Wada I., Kobayashi Y., Nagaoka K., Kushihara Y., Ushiku T., Seto Y, & Kakimi K. (2022). Selection of RNA-based evaluation methods for tumor microenvironment by comparing with histochemical and flow cytometric analyses in gastric cancer. Scientific Reports, 12, 8576.

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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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