All specimens were pathologically reviewed and assessed retrospectively by two investigators (K.I and M.K). Maximum tumor size was determined based on H.E. slides and neuroendocrine differentiation was assessed by positive immunohistochemical staining for chromogranin A (diluted 1:200, clone DAK-A3, Dako, Glostrup, Denmark), synaptophysin (ready to use, DAK-A3, Dako, Glostrup, Denmark), and CD56 in all cases (diluted 1:50, clone 123C3, Dako, Glostrup, Denmark). Tumor specimens were considered positive for neuroendocrine markers if more than 5% of tumor cells were stained. All tumors are positive for at least two of three neuroendocrine markers. Elastica and D2–40 staining was used in all cases to assess lymph-vascular invasion (diluted 1:200, clone D2–40, Acris, Herford, Germany). The Ki-67 and mitotic index was evaluated, and grading was performed according to the WHO classification 201015 . Lymphatic invasion and venous invasion were assessed as reported previously27 (link). Lymph node metastasis was confirmed histologically using surgical specimens, and distant metastases were evaluated either radiologically or histologically.
Dak a3
Manufactured by Agilent Technologies
Sourced in Denmark, China, United Kingdom, United States
The DAK-A3 is a broadband dielectric analyzer from Agilent Technologies. It is designed to measure the dielectric properties of materials over a wide frequency range.
Automatically generated - may contain errors
Lab products found in correlation
Clone 123c3, by Agilent Technologies (2 mentions)
Clone dak a3, by Agilent Technologies (1 mentions)
Mib 1, by Agilent Technologies (1 mentions)
Ck ae1 ae3, by Agilent Technologies (1 mentions)
Vimentin v9, by Agilent Technologies (1 mentions)
Dak synap, by Agilent Technologies (1 mentions)
Omnis automated staining platform, by Agilent Technologies (1 mentions)
Sp347, by Roche (1 mentions)
D6h2l, by Cell Signaling Technology (1 mentions)
Ncl l pgr 312, by Leica Biosystems (1 mentions)
Iview dab detection kit, by Roche (1 mentions)
Benchmark autostainer, by Roche (1 mentions)
Dako autostainer, by Agilent Technologies (1 mentions)
Ventana benchmark ultra, by Roche (1 mentions)
8 protocols using dak a3
1
Neuroendocrine Tumor Histopathological Assessment
2
Comprehensive Immunohistochemical Analysis of NMC
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Available tissue sections were used for immunohistochemistry (IHC) analyses of these 5 NMC cases. Surgical tissue sections were sectioned at 4 μm thickness and then stained with primary antibodies specific for NUT (ab122649, 1 : 500, Abcam, UK), CK (AE1/AE3, prediluted; Dako, Denmark), vimentin (V9, prediluted; Dako), CK7 (EP16, 1 : 200, ZSGB‐BIO, China), SMA (1A4, 1 : 200, ZSGB-BIO), CD34 (QBEND, prediluted, Dako), p63 (ZM-0406, prediluted, ZSGB‐BIO), p40 (ZA-0483, 1 : 100, ZSGB‐BIO), Syn (DAK-SYNAP, prediluted, Dako), CD56 (123C3, prediluted, Dako), CgA (DAK-A3, prediluted, Dako), and Ki-67 (MIB-1, prediluted, Dako). A Dako Omnis automated staining platform was used for the IHC staining of these formalin-fixed paraffin-embedded whole tissue sections. Appropriate positive controls were used for all assays.
3
Immunohistochemical Analysis of Lung Tumors
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Lobectomy and biopsy specimens were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections of 4-μm thickness were stained with hematoxylin and eosin and immunohistochemistry was conducted for CD56 (NCAM, Clone 1B6, Novocastra Leica Biosystems, Newcastle, UK), chromogranin A (DAK-A3, Dako, Glostrup, Denmark), synaptophysin (27G12, Leica Biosystems, Nussloch, Germany), TTF-1 (NKX2-1, Clone 8G7G3/1, Dako), c-MYC (Y69, Ventana Medical Systems Inc., Tucson, AZ, USA), DLL3 (SP347, Ventana Medical Systems Inc.), and RB protein (13A10, Leica Biosystems) using an autoimmunostainer (Ventana XT System Benchmark; Ventana Medical Systems Inc.).
4
Immunohistochemical Analysis of Tumor Samples
5
Immunohistochemical Labeling Protocol
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Immunohistochemical labeling was performed at the immunohistochemical laboratory of the Department of Pathology, Asan Medical Center. In brief, 4-mm-thick tissue sections were deparaffinized and hydrated in xylene and serially diluted in ethanol. Endogenous peroxidase was blocked by incubation in 3% H2O2 for 10 minutes, and then heat-induced antigen retrieval was performed. Primary antibodies were used with a Benchmark autostainer (Ventana Medical Systems, Tucson, AZ, USA) in accordance with the manufacturer’s protocol. Sections were incubated at room temperature for 32 minutes in primary antibodies for PR (1:200, NCL-L-PGR-312, Novocastra, Newcastle upon Tyne, UK), synaptophysin (1:200, DiNona, Seoul, Korea), chromogranin (1:200, DAK-A3, DakoCytomation, Glostrup, Denmark), and Ki-67 (1:100, 7B11, Zymed, San Francisco, CA, USA). The sections were then labeled with an automated immunostaining system and processed with an iView DAB detection kit (Benchmark XT, Ventana Medical Systems). Immunostained sections were lightly counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene. Immunoreactivity was interpreted by light microscopic examination and independently evaluated by two pathologists, coauthors of this study (S.J.K. and S.M.H.), who were blind to the clinicopathologic information.
6
Immunohistochemical Profiling of Neuroendocrine Tumors
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Tissue sections including whole slide of the representative block and tissue microarrays were deparaffinized and hydrated, and endogenous peroxidase activity was blocked. Antigen retrieval was achieved using Dako Target Retrieval Solution, High PH (Dako, Produktionsvej, DK; 50×) in a PT Link set at 98 °C for 25 min. Afterwards the tissue sections were incubated with an anti-SOX11 mouse monoclonal antibody (MRQ-58, Cell MARQU, USA), anti-INSM1 (A-8, Santa Cruz Biotechnology, USA), SYN (MRQ-40, Cell MARQU, USA), CGA (DAK-A3, DAKO, USA), CD56 (123C3D5, Cell MARQU, USA) for 30 min at room temperature, and detection was achieved using an enzyme-conjugated polymer complex adapted for automatic stainers from DAKO (Dako, Dako Autostainer, Produktionsvej, DK). Tumor cells of mantle cell lymphoma were used as a positive control. Paratumoral mature mesenchymal or epithelial cells served as negative controls. All immunostains were recorded for intensity of reactivity (0, none; 1+, weak; 2+, moderate; 3+ strong) and percentage of positive neoplastic cells. Positive immunostaining for CD56, SYN and CGA required 10% or more cells with an intensity of at least 2+ on cytoplasmic or membranous localization [21 (link)]. For SOX11 and INSM1, at least 1+ nuclear staining in > 10% of tumor cells was considered positive [10 (link)].
7
Neuroendocrine Marker Analysis in FFPE Tumor Samples
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Formalin‐fixed, paraffin‐embedded tumor samples were analyzed by immunohistochemistry (IHC) using antibodies to CD56 (Dako, clone 123C3, 1:100), Syn (Dako, clone DAK-SYNAP, 1:50), INSM1 (Santa Cruz Biotechnology, clone A-8, 1:400), and CgA (Dako, clone DAK-A3, 1:100). The experimental procedure was performed by following the manufacturer's instructions, and IHC stains were evaluated by two pathologists. Expression of each neuroendocrine marker was semi-quantified using H-scores (range 0–300), which incorporate the staining intensity (range 0–3+) and the percentage of positively-stained tumor cells (range 0–100%).
8
Immunohistochemical Analysis of Tumor Markers
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Immunohistochemical analysis (IHC) was performed using a Ventana BenchMark Ultra autostainer (Ventana Medical Systems Inc, 1910 Innovation Park Drive, Tucson, Arizona, USA) according to the manufacturer's instructions. Whole tissue consecutive, 3-µm cuts were made from embedded tissues. The following antibodies were used for analysis: desmin (D33, mouse monoclonal, Dako, dilution 1:50), myogenin (F5D, monoclonal mouse, Dako, dilution 1:50), synaptophysin (rabbit polyclonal, ThermoScientific, dilution 1:350), and chromogranin A (DAK-A3, monoclonal mouse, Dako, 1:400). Expression of these four markers was scored as strong, moderate or weak and diffuse or focal. Diverse other markers were used in a case-to-case basis according to the most pertinent differential diagnostic considerations at time of initial biopsy assessment.
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