Immunohistochemistry was performed on paraffin-embedded tissues sections (3-4 μm-thick) using the following primary antibodies: ER-α (clone 6F11, monoclonal; Novocastra, Newcastle-Upon-Tyne, UK), PgR (clone PgR636, monoclonal; DakoCytomation, Glostrup, Denmark) and HER2 (A0485, polyclonal; DakoCytomation). Diaminobenzidine (Dako) was used as the chromogen. ER and PgR expression was considered positive when at least 10% of invasive tumoral cells exhibited nuclear staining, regardless of intensity. For HER2, the immunohistochemical score was assigned according to the Herceptest scoring system [19 (link)].
Er α clone 6f11
Manufactured by Leica
Sourced in United Kingdom
ER-α (clone 6F11) is a monoclonal antibody that recognizes the estrogen receptor alpha (ER-α) protein. It is designed for use in immunohistochemical (IHC) applications to detect the presence and localization of ER-α in various tissue samples.
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Lab products found in correlation
Pgr clone pgr636, by Agilent Technologies (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Dab solution, by Agilent Technologies (1 mentions)
Dab sparkle enhancer, by Biocare Medical (1 mentions)
Cytoseal, by Avantor (1 mentions)
P her2, by Cell Signaling Technology (1 mentions)
Novolink polymer detection system, by Leica (1 mentions)
4 protocols using er α clone 6f11
1
Immunohistochemical Biomarker Evaluation
2
Immunohistochemical Analysis of Breast Cancer Biomarkers
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Sections (4 μm thick) were cut from formalin-fixed, paraffin-embedded tissues derived from whole tissue sections from representative blocks for each case. These sections were cut, dried, deparaffinised and rehydrated according to standard procedures. All sections were subjected to heat-induced antigen retrieval in citrate buffer (pH 6.1). Antibodies against ERα (clone 6 F11, 1:200; Novocastra, Milton Keynes, UK), ERBB2 (clone CB11, 1:1,000; Novocastra) and MUC1 (clone DF3, 1:100; Bio SB, Goleta, CA, USA) were incubated for 1 hour at room temperature. Staining for horseradish peroxidase antimouse and antirabbit immunoglobulin G was detected with the universal VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine (Dako A/S, Glostrup, Denmark) as chromogen. Internal and external controls were included for each antibody. The American Society of Clinical Oncology–defined cutoffs were used to determine whether cases were positive for ER (≥1%) or for ERBB2 (≥30%) by complete and intense membranous staining [16 (link),17 (link)]. MUC1 protein localisation (apical or cytoplasm) was also determined.
3
Quantifying p-HER2 and ER+ in Xenograft Tissues
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IHC staining was executed with the help of the Pathology Core at the Lester and Sue Smith Breast Center, Baylor College of Medicine. Tissue sections were incubated at 58°C overnight in a dry slide incubator and deparaffinized in xylene and graded alcohol washes. Antigen retrieval was performed in 0.1 M Tris-HCl pH 9.0 (p-HER2 and ERα), followed by quenching in 3% H2O2. The following antibodies were used to stain for 1 hour at room temperature: ERα (clone 6F11, Novocastra, 1:200) and p-HER2 (Cell Signaling, 2243L, 1:50). After washing in TBS, EnVision labeled polymer-HRP anti-mouse or anti-rabbit antibodies (Dako) were added for 30 minutes at room temperature. Slides were washed with TBS and then developed with DAB+ solution (Dako) and DAB sparkle enhancer (Biocare). After washing in TBS, slides were counterstained with hematoxylin, dehydrated, and cleared before coverslipping with Cytoseal (VWR). p-HER2 and ER+ stained cells were quantified in lung/ovary sections of L755S fat pad (FP) xenografts and ovary sections from HER2 WT and HER2 S310F or L755S MIND xenograft mice.
4
Histopathological and Immunohistochemical Characterization of Mammary Tumors
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The fixed tissues were trimmed, processed and embedded in paraffin and 2-μm-thick sections were stained with haematoxylin and eosin (H&E) for histopathological evaluation by three independent pathologists (A. Alvarado, R.M. Gil da Costa, C. Lopes). The mammary tumours were classified and categorized considering the predominant histological patterns in each mammary tumour according to Russo and Russo (Russo & Russo 2000) . Immunohistochemistry was performed on 2-μm-thick sections, using the Novolink Polymer Detection System (Leica Biosystems, Newcastle, UK) according to the instructions provided by the manufacturer. Both primary antibodies for ER α (clone 6F11, Novocastra, Newcastle, UK) and PR (clone SP2, Abcam, Cambridge, UK) were diluted in phosphate-buffered saline (PBS) at 1:50 and incubated for 1.5 h at room temperature and 16 h (overnight) at 4°C respectively. The immunoexpression of ER α and PR was evaluated in primary tumours (in each histological pattern) and their metastases, counting the number of immunostained cells (cells with punctual nuclear labelling) in at least 500 neoplastic cells. Results were expressed as the percentage of immunopositive neoplastic cells. Normal rat mammary tissues incubated with and without the primary antibody were used as positive and negative controls respectively.
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