FFPE tissue was analyzed by IHC using Anti-Her-2 (Polyclonal, DAKO) as per standard procedures and the results were analyzed as per ASCO-CAP guidelines.
Anti her2
Manufactured by Agilent Technologies
Sourced in United States, Denmark
Anti-HER2 is a laboratory reagent used for the detection and quantification of the HER2 protein in biological samples. It is designed to support research and development activities related to HER2-targeted therapies.
Automatically generated - may contain errors
Lab products found in correlation
Anti ki67, by Agilent Technologies (1 mentions)
Benchmark xt, by Roche (1 mentions)
Anti ck5 6, by Agilent Technologies (1 mentions)
Anti pr, by Agilent Technologies (1 mentions)
Sector imager 2400 reader, by Mesoscale (1 mentions)
Anti epcam, by Abcam (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab reagent, by Agilent Technologies (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti pr clone pgr 636, by Agilent Technologies (1 mentions)
Laser confocal microscope, by Nikon (1 mentions)
Fluoro care anti fade mounting medium, by Biocare Medical (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Af2659, by R&D Systems (1 mentions)
Lsab kit, by Agilent Technologies (1 mentions)
Anti er antibody, by Agilent Technologies (1 mentions)
Anti pr antibody, by Agilent Technologies (1 mentions)
Anti muc2, by Leica (1 mentions)
Anti smad4, by Santa Cruz Biotechnology (1 mentions)
11 protocols using anti her2
1
IHC-Based HER2 Evaluation in FFPE Tissue
2
Molecular Subtyping of Salivary Carcinomas
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A total of 22 out of 29 SeCs were classified into 5 molecular subtypes in a similar way to breast cancer, as described in our previous study (luminal 1, luminal 2, HER2, all-negative, and core basal) (Table S2 ) [24 (link)]. The remaining 7 cases had no available tissue for IHC study. An automated immunostainer (Ventana BenchMark XT, Tucson, AZ, USA) was used to perform the IHC for anti-HER2 (Dako, Carpinteria, CA, USA), antiandrogen receptor (anti-AR; Thermo Scientific, Carlsbad, CA, USA), anti-ER (Dako), anti-PR (Dako), anti-CK5/6 (Dako), and anti-Ki67 (Dako) antibodies. The interpretation of HER2 IHC results was based on the 2013 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines [34 (link)]. Finally, scores of 2+ and 3+ were considered positive for HER2. For hormone receptors (HRs), including AR, ER, and PR, it was considered positive when ≥1% of the tumor cell nuclei were stained. CK5/6 was scored when ≥50% of the tumor cells were strongly immunoreactive [24 (link)].
3
Quantitative Analysis of HER-2 Expression
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In order to quantitate HER-2 expression values, we performed well-based reverse phase protein array (RPPA) as previously reported [13 (link)]. Briefly, extracted proteins (400 ng) were applied onto 96-well Multi-Spot™ plates (Meso Scale Discovery, Gaithersburg, MD, MA2400 96 HB Plate), the plate was allowed to dry at RT, and if needed, further incubated at 37°C for 30 min. The antigen-coated plates were pre-incubated with 5% BSA in PBST for 60 min at RT before primary antibody incubation. Anti-HER-2 (DAKO) and anti-GAPDH (Calbiochem) were diluted 1:1000 and 1:5000 with 5% BSA in PBST, followed overnight incubation at 4°C. After washing with PBST, the plates were incubated for 90 min with goat anti-rabbit or mouse SULFO-TAG™ antibodies (Meso Scale Discovery) at a dilution of 1:2000. The plates were washed three times with PBST. MSD-T read buffer was added to the plates and signals were detected using Sector Imager 2400 reader (Meso Scale Discovery). BSA coated wells were included on each plate as a control of non-specific binding. HER-2 expression signal was normalized with the value of GAPDH.
4
Immunohistochemical Profiling of Breast Markers
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For immunohistochemistry, formalin-fixed and paraffin-embedded tissues were sectioned to 4 μm thickness, deparaffinized, rehydrated, and prepared for antigen retrieval. The slides were then incubated at room temperature for 1 h with the following antibodies at appropriate dilution: anti-ER (6F11, Novocastra), anti-PR (1A6, Novocastra), anti-HER2 (polyclone, DAKO), anti-cytokeratin 18 (CK 18) (DC-10, BioGenex), anti-cytokeratin 19 (CK19) (RCK108, BioGenex), anti-CK 5/6 (D5/16 B4, DAKO), anti-EGFR (EGFR.25, Novocastra), anti-E-cadherin (36B5, Novocastra), and anti-EpCAM (polyclonal, Abcam). After incubation, the slides were washed thrice with PBS, incubated with horseradish peroxidase (HRP) conjugated antibody (Zymed) at room temperature for 10 min, and developed by adding 3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent (DAKO) as the chromogen and hematoxylin as the counterstain. Expression of ER and PR was rated using Allred score. All specimens were independently reviewed by two pathologists blinded to the clinical origin of the specimens.
5
Detecting HER2 Protein in Carcinoma Cells
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Proteins from canine mammary carcinoma cell lines CypP and CypM [27 (link)] and human ovarian carcinoma cell line SKOV3 were extracted in lysis buffer (1% Triton X-100, 10% glycerol, 50 mM Tris, 150 mM sodium chloride, 2 mM EDTA, pH 8.0, and 2 mM magnesium chloride) containing protease inhibitor cocktail (P8340, Sigma Aldrich, Darmstadt, Germany). An amount of 20 micrograms of total proteins was separated in SDS-PAGE (10% or 15%) and transferred onto a PVDF membrane. After washing, membranes were incubated in TBS/BSA 10% (bovine serum albumin) at room temperature for 1 h and then incubated overnight at 4 °C with a polyclonal antibody anti-HER2 (A0485, Dako, Glostrup, Denmark). After incubation, membranes were washed in TBS/Tween 0.01%, and specific bands were revealed by horseradish peroxidase secondary antibodies.
6
Immunohistochemical Profiling of Breast Tumors
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Tissue specimens were fixed in 10% formalin and subsequently embedded in paraffin. Then, 2-μm-thick paraffin-embedded tissue sections were mounted onto glass slides. All slides were stored for two days at 58 °C in a drying chamber, subsequently deparaffinized using xylene and hydrated with ethanol. Human and corresponding xenograft tumor tissues were stained using the following antibodies: Anti-ER (clone EP1, code IR084, Dako, Santa Clara, CA, USA), anti-PR (clone PgR 636, code IR068, Dako, Santa Clara, CA, USA), anti-HER2 (code A0485, Dako, Santa Clara, CA, USA). Counterstaining was performed with hemalum before adding a coverslip. As positive controls, a patient-derived physiological mammary gland was used for ER and PR staining. For HER2, tissue specimens from HER2-positive breast cancer patients (score 3) [88 (link)] were included. TNBC was defined as ER-, PR-, and HER2-negative (score < 2) [89 (link)].
7
Immunofluorescence Analysis of MUC13 and Oncoproteins
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Immunofluorescence staining was performed to determine the effect of miR-145 transfection on the protein level of MUC13 and other related key oncogenic proteins. Cells were grown at a low density for 24h on the chamber slides (Nalge Nunc Int) and processed for immunofluorescence as described [28 (link)]. Cells were incubated with anti-MUC13 MAb (clone PPZ020) or anti-HER2 (catalog number A0485; DAKO), anti-p53 (catalog number 2527; Cell Signaling) followed by an incubation with species specific Alexa Fluor 488 (catalog number A11029; Invitrogen) or Alexa Fluor 568 (catalog number A11036; Invitrogen) secondary antibodies and were mounted in Fluoro Care Anti-Fade mounting medium (BioCare Medical, CA, USA). The cells were examined under a laser confocal microscope (Nikon Corporation).
8
Nectin-4 Expression in Breast Cancer
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Tissue microarrays were constructed by extracting 2-mm diameter cores of histologically confirmed invasive breast carcinoma areas, as previously described.30 (link) Five-micrometer tissue sections were cut and stained using the purified goat polyclonal antibody raised against the recombinant human Nectin-4 extracellular domain (1:60 dilution, 60 min, AF2659, R&D Systems Inc., Minneapolis, MN, USA). Whole sections of non-neoplastic breast tissues from 30 patients were also stained. As positive controls of Nectin-4 expression, skin and nipple sections were used.18 (link) Antigen retrieval was performed by microwave treatment at 750 W for 10 min in 1 M urea buffer (pH 8.0). The LSAB kit (K0679, Dako, Glostrup, Denmark) was used for signal amplification. In control sections, the specific primary antibody was replaced with isotype-matched immunoglobulins. The following antibodies were used for the identification of tumor subtypes, as previously detailed:31 (link) the anti-ER-α MoAb 6F11 (Novocastra, Menarini, Florence, Italy), the anti-PR MoAb 1A6 (Menarini), the anti-Ki67 MoAb MIB-1 (Dako) and the anti-HER-2 (Herceptest, Dako). Immunohistochemical analysis was done by two pathologists (MP, RL) by consensus without knowledge of the clinicopathological information.
9
Breast Cancer Immunohistochemical Profiling
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Immunohistochemistry (IHC) for PR, ER, HER2/neu and Ki-67 was accomplished on FFPE tissue segments as part of the routine clinical assessment of these cases using anti-ER antibody (DAKO, Denmark),anti-ER antibody (DAKO, Denmark), anti-HER2 (1:400 to 1:600, DAKO, Denmark), anti-PR antibody (DAKO, Denmark) and Ki-67 (DAKO, Denmark) with Visualize system for detection. Lobular and ductal normal areas of breast were as control for ER, PR and HER2 IHC whereas appendix tissue was set as control for Ki-67. Olympus (Model U-DO3) was used for microscopy.
10
Immunophenotypic Evaluation of Pancreatobiliary Neoplasms
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Intraductal lesions and invasive carcinomas were evaluated separately by immunohistochemistry using the following antibodies that have been shown to be differentially expressed in pancreatobiliary neoplasms: 9,28-30 anti-MUC1 (Novocastra, Newcastle upon Tyne, UK; 1:160), anti-MUC2 (Novocastra; 1:100), anti-MUC5AC (Leica Microsystems, Bannockburn, IL, USA; 1:200), and anti-MUC6 (Leica Microsystems; 1:80), anti-EGFR (Dako, Carpinteria, CA, USA; RTU ready-to-use), anti-TP53 (Dako; 1:80), anti-HER2 (Dako; 1:80), anti-SMAD4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:400), anti-CDKN2A/p16 (CINtec, Ventana Medical Systems, Tucson, AZ, USA; ready-to-use), and anti-CDX2 (Biogenex, San Ramon, CA, USA; 1:200). All Modern Pathology (2015) 28, 1249-1264 immunolabeling was run on an automated immunostainer according to the company's protocols for open procedures with slight modifications. The immunohistochemical labeling of the antibodies listed above was evaluated as described before. 9
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