M7047

Manufactured by Agilent Technologies

Sourced in Denmark

The M7047 is a laboratory equipment product from Agilent Technologies. It is designed to perform specific tasks within a laboratory environment. The core function of the M7047 is to provide precise and reliable measurements, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

12 protocols using m7047

Immunohistochemical Characterization of Mammary Tumors

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Tumors were histopathologically characterized on HE-stained sections following the routine method for histological description of neoplasms [5 (link)]. Immunohistochemical characterization of estrogen and progesterone receptors (ER, Ref. M7047, Dako; PR, Ref. 790-2223, Ventana, Oro Valley, AZ, USA) and human epidermal receptor-2 (HER-2, Ref. A0485, Dako, Santa Clara, CA, USA) was performed. Paraffin sections were placed in a PT module, heated for 20 min at 95 °C, and cooled down to 60 °C. Then, slides were rinsed in warm tap water and placed in an automatic immunostainer device (Lab Vision Corp., Fremont, CA, USA) for immunohistochemistry using a peroxidase detection system. After immunostaining, the slides were counterstained with hematoxylin and permanently mounted with Depex. Corresponding negative control slides were prepared by replacing the primary antibody with nonreactive antibody. Slides from human and canine mammary tumors with previously demonstrated reactivity to the primary antibody and tissue internal controls were used as positive controls [5 (link)].
For estrogen receptor, progesterone receptor, and HER-2 evaluation, 3+ positive scoring was considered, following the recommended guidelines of the American Society of Cancer Oncology (ASCO).

Caceres S., Alonso-Diez A., Crespo B., Peña L., Illera M.J., Silvan G., de Andres P.J, & Illera J.C. (2021). Tumor Growth Progression in Ectopic and Orthotopic Xenografts from Inflammatory Breast Cancer Cell Lines. Veterinary Sciences, 8(9), 194.

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Evaluation of Breast Cancer Biomarkers

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The ER, PgR and HER2 expressions were evaluated using the immunohistochemically stained slides retrieved from the archives of the department. For IHC staining of all breast cancers to assess the ER, PgR and HER2 expression, primary monoclonal mouse antihuman estrogen receptor α clone 1D5 (Dako-M7047), monoclonal mouse antihuman progesterone receptor (Dako- M3569) and polyclonal rabbit antihuman c-erbB-2 oncoprotein (Dako-A0485) respectively have been used with the secondary antibody (Dako Real EnVision™). The Allred score was used to assess ER and PgR status. The UK recommendations were used for the assessment of HER2 expression [14 (link)]. IHC assessment was done by a single investigator in order to eliminate inter-observer bias. Patients who had no staining for ER, PgR and a score of 0 or + 1 for HER2 were considered as triple negative (TNBC). Breast cancers were scored as ER/PgR positive if the total Allred score for ER/PgR was > 2/8. A score of + 3 was considered positive for HER2 over expression. Patients who had + 2 for HER2 and found to be positive by FISH were also included as HER2 positive.

Peiris H., Mudduwa L., Thalagala N, & Jayatilake K. (2018). Validity of St Gallen risk categories in prognostication of breast cancer patients in Southern Sri Lanka. BMC Women's Health, 18, 30.

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Immunohistochemical detection of ERα, PR, and Ki67

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Primary antibodies to detect ERα (ID5 1:300; DAKO #M7047, Glostrup, Denmark), PR (1:1000, Leica #NCL-PgR-AB, Wetzlar, Germany) or Ki67 (MIB1 1:400; DAKO #M7240 Glostrup, Denmark) were used in conjunction with a 1:400 dilution of a biotinylated anti-mouse secondary antibody for 30 min (DAKO #E0433, Glostrup, Denmark) followed by incubation with HRP-conjugated streptavidin (DAKO #P0397, Glostrup, Denmark). Visualization of immunostaining was performed using 3,3-Diaminobenzidine (Sigma #D9015), as previously described^{36 (link)}.

Mohammed H., Russell I.A., Stark R., Rueda O.M., Hickey T.E., Tarulli G.A., Serandour A.A., Birrell S.N., Bruna A., Saadi A., Menon S., Hadfield J., Pugh M., Raj G.V., Brown G.D., D’Santos C., Robinson J.L., Silva G., Launchbury R., Perou C.M., Stingl J., Caldas C., Tilley W.D, & Carroll J.S. (2015). Progesterone receptor modulates estrogen receptor-α action in breast cancer. Nature, 523(7560), 313-317.

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Immunohistochemical Analysis of Tissue Markers

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Dewaxed hydrated paraffin-embedded tissue sections were immersed in 3% H₂O₂ and 100% methanol for 30 min at room temperature to quench endogenous peroxidase, and then the sections were blocked with 10% normal goat (for anti-HDAC3, Ki-67, ESR1, PGR, vimentin, and COUP-TFII antibodies) or rabbit (for anti-COL1 antibody) serum in PBS (pH 7.5) and incubated with anti-HDAC3 (1:1000 dilution, sc11417, Santa Cruz Biotechnology), anti–Ki-67 (1:1000 dilution, ab15580, Abcam), anti-ESR1 (1:500 dilution, M7047, DAKO), anti-PGR (1:500 dilution, A0098, DAKO), anti-vimentin (1:10,000 dilution, ab92547, Abcam), anti–COUP-TFII (1:500 dilution, PP-H7147–00, Perseus Proteomics), or anti-COL1 (1:1000 dilution, 1310–01, SouthernBiotech) antibodies overnight at 4°C. On the following day, the sections were incubated with secondary antibody conjugated to horseradish peroxidase (Vector Laboratories) for 1 hour at room temperature. Immunoreactivity was detected using diaminobenzidine (DAB; Vector Laboratories) and analyzed using microscopy software from NIS Elements Inc. (Nikon). The H-score was calculated using the following equation: H-score = Σ Pi (i), where i is the intensity of staining with a value of 1, 2, or 3 (weak, moderate, or strong, respectively) and Pi is the percentage of stained cells for each intensity, varying from 0 to 100%.

Kim T.H., Yoo J.Y., Choi K.C., Shin J.H., Leach R.E., Fazleabas A.T., Young S.L., Lessey B.A., Yoon H.G, & Jeong J.W. (2019). Loss of HDAC3 results in nonreceptive endometrium and female infertility. Science translational medicine, 11(474), eaaf7533.

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Immunohistochemical Identification of Estrogen Receptors and HER2 in Cytology Specimens

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Monoclonal antibody 1D5 was used to identify estrogen receptors (ERs) in patients (M7047; DakoCytomation, Carpinteria, CA). For cytologic smears, the immunohіstochemical procedure for ERs was identical to the procedure used for the histologic slides and did not require destaіning of the smears. In cytologic specimens, no immunohіstochemical analysis for progesterone receptors (PRs) was carried out. HER2 staining was performed using the Ventana іVIEW DAB Detection Kit. The staining procedure using this kit is based on the indirect biotin streptavidin system. The heat antigen recovery protocol was used for paraffin-embedded sections as recommended by the manufacturer. The primary antibody, the rabbit monoclonal Ventana I‐V primary antibody (4B5), was used for PATHWAY (Roche Diagnostics) anti-HER 2/neu. The main antibody was the primary antibody.

Alkasaby M.K., Abd El-Fattah A.I., Ibrahim I.H, & Abd El-Samie H.S. (2020). Polymorphism of XRCC3 in Egyptian Breast Cancer Patients. Pharmacogenomics and Personalized Medicine, 13, 273-282.

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Immunohistochemical detection of ERα, PR, and Ki67

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Immunohistochemical Validation of Breast Cancer Markers

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Immunostainings were performed on 4 μm paraffin-embedded tissue sections. The sections were deparaffinized and rinsed with 10 mM Tris–HCl (pH 7.4) and 150 mM sodium chloride. Peroxidase was quenched with methanol and 3% hydrogen peroxide. Slides were placed in 10 mM citrate buffer (pH 6.0) at 100 °C for 20 min in a pressurized heating chamber^{53 (link)}. Tissue sections were incubated with the antibodies against GPNMB (1:200; AI12434, Abgent), E-cadherin (1:200; Clone GM016, Genemed), ZO-1 (1:200; 61–7300, Invitrogen), N-cadherin (1:20; NCL-L-N-Cad, Leica Biosystems) and vimentin (1:200; clone V9, Leica Biosystems) for 1 h at room temperature and then washed with phosphor-buffered saline. Bound antibodies were detected using the EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse kit (Dako, Denmark). The slides were counterstained with hematoxylin. For negative controls, above IHC steps were performed without primary antibody. Mouse monoclonal antibodies were used to detected ER (M7047, Dako), PR (M3569, Dako), and erbB-2 (28-0003z, Invitrogen). Tissues were identified as ER or PR positive breast cancer if more than 10% nuclei were stained and as Her2-positive breast cancer if 3 + Her2 expression was scored.

Huang Y.H., Chu P.Y., Chen J.L., Huang C.T., Huang C.C., Tsai Y., Wang Y.L., Lien P.J., Tseng L.M, & Liu C.Y. (2021). Expression pattern and prognostic impact of glycoprotein non-metastatic B (GPNMB) in triple-negative breast cancer. Scientific Reports, 11, 12171.

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Immunohistochemical Analysis of Tumor Samples

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Tumor tissue was collected and immediately fixed in 10% neutral-buffered formalin at 4 °C overnight before dehydration and paraffin embedding. Antibodies used for immunohistochemistry were anti-ER (M7047, 1:300, Agilent) and anti-Ki-67 (M7240, 1:400, Agilent). Primary antibodies were detected using biotinylated IgG secondary antibodies (Agilent, 1:400), using streptavidin-HRP (Agilent) for amplification of signal followed by the addition of 3,3′-diaminobenzidine (Sigma) substrate. Images were scanned using Leica Aperio Slide Scanner (Leica Biosystems) and analyzed using QuPath software to differentiate tumor tissue from stroma and necrosis, and to quantify Ki-67 positivity in tumor tissue.

Achinger-Kawecka J., Stirzaker C., Portman N., Campbell E., Chia K.M., Du Q., Laven-Law G., Nair S.S., Yong A., Wilkinson A., Clifton S., Milioli H.H., Alexandrou S., Caldon C.E., Song J., Khoury A., Meyer B., Chen W., Pidsley R., Qu W., Gee J.M., Schmitt A., Wong E.S., Hickey T.E., Lim E, & Clark S.J. (2024). The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer. Nature Structural & Molecular Biology, 31(3), 498-512.

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Immunohistochemical Evaluation of PRLR and ER-α

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Immunohistochemical analysis was performed as previously described [8 (link)]. Anti-PRLR (B6.2) antibody (ab74608; Abcam) [15 ] and antiestrogen receptor alpha (ER-α) antibody (M7047; Agilent) [16 ] were used for staining the endometrium tissues. The immunoreactive score was calculated by multiplying the optical staining intensity (graded as 0 = no, 1 = weak, 2 = moderate, and 3 = strong staining) and the percentage of positive stained cells (0 = no staining, 1 = < 10% of the cells, 2 = 11–50% of the cells, 3 = 51–80% of the cells, and 4 = >81% of the cells) for an evaluation of the expression of PRLR and ER-α as previously described [17 (link)].
The immunostained slides were evaluated independently by two of the authors who were not informed about the clinical backgrounds. An average score of two authors was used for comparison of PRLR and ER-α.

Yamaguchi M., Erdenebaatar C., Saito F., Honda R., Ohba T., Kyo S., Tashiro H, & Katabuchi H. (2019). Prolactin Enhances the Proliferation of Proliferative Endometrial Glandular Cells and Endometrial Cancer Cells. Journal of the Endocrine Society, 4(2), bvz029.

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Immunohistochemical Profiling of Tumor Samples

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Tumour tissue was harvested and immediately fixed in 10% neutral buffered formalin at 4 °C overnight before dehydration and paraffin embedding. Antibodies used for IHC were anti-ER (M7047, 1:300, Agilent) and anti-Ki67 (M7240, 1:400, Agilent). Primary antibodies were detected using biotinylated IgG secondary antibodies (Agilent, 1:400), using streptavidin-HRP (Agilent) for amplification of signal followed by the addition of 3,3′-diaminobenzidine (Sigma) substrate. Images were scanned using Leica Aperio Slide Scanner (Leica Biosystems) and analysed using QuPath software to differentiate tumour tissue from stroma and necrosis, and to quantify Ki-67 positivity in tumour tissue.

Portman N., Milioli H.H., Alexandrou S., Coulson R., Yong A., Fernandez K.J., Chia K.M., Halilovic E., Segara D., Parker A., Haupt S., Haupt Y., Tilley W.D., Swarbrick A., Caldon C.E, & Lim E. (2020). MDM2 inhibition in combination with endocrine therapy and CDK4/6 inhibition for the treatment of ER-positive breast cancer. Breast Cancer Research : BCR, 22, 87.

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