Clone 123c3

Manufactured by Agilent Technologies

Sourced in Denmark

The Clone 123C3 is a laboratory equipment product manufactured by Agilent Technologies. It is designed to perform core functions related to DNA cloning and analysis. The detailed specifications and intended use of this product are not available.

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CD56 (clone 123C3, (6 citations) Mouse anti-CD56 (clone 123C3, (2 citations)

9 protocols using clone 123c3

Neuroendocrine Tumor Histopathological Assessment

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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 previously^{27 (link)}. Lymph node metastasis was confirmed histologically using surgical specimens, and distant metastases were evaluated either radiologically or histologically.

Kojima M., Chen Y., Ikeda K., Tsukada Y., Takahashi D., Kawano S., Amemiya K., Ito M., Ohki R, & Ochiai A. (2019). Recommendation of long-term and systemic management according to the risk factors in rectal NETs patients. Scientific Reports, 9, 2404.

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Immunohistochemical Profiling of Tumor-Infiltrating Lymphocytes

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Tissue microarrays were constructed as described previously.26 (link) Immunohistochemical (IHC) staining was performed using mouse anti-human CD3 (Abcam, clone SP7), mouse anti-human CD8 (Dako, clone C8/144B), rabbit anti-human TIGIT (Abcam, clone BLR047F), and mouse anti-human CD56 (Dako, clone 123C3). TIL and IHC expression scores were determined by a blinded pathologist (MAD). TIL scores were calculated on H&E-stained slides as follows: 3 (>20 TIL/high-power field (hpf)), 2 (11–20 TIL/hpf), 1 (1–10 TIL/hpf), 0 (<1 TIL/hpf). IHC expression for CD3, CD8, CD56, and TIGIT was calculated as follows to derive an H-score: [1x (% 1+cells)+2x(% 2+cells)+3x(% 3+cells)] where 1+, 2+, and 3+ represent weak, moderate, and strong stain intensity, respectively. Final TIL and IHC expression scores were averaged when more than one consecutive section was present for scoring. PD-L1 staining of tumor samples was performed using a CLIA-certified laboratory.

Judge S.J., Darrow M.A., Thorpe S.W., Gingrich A.A., O'Donnell E.F., Bellini A.R., Sturgill I.R., Vick L.V., Dunai C., Stoffel K.M., Lyu Y., Chen S., Cho M., Rebhun R.B., Monjazeb A.M., Murphy W.J, & Canter R.J. (2020). Analysis of tumor-infiltrating NK and T cells highlights IL-15 stimulation and TIGIT blockade as a combination immunotherapy strategy for soft tissue sarcomas. Journal for Immunotherapy of Cancer, 8(2), e001355.

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Immunohistochemical Evaluation of Thyroid Nodules

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The primary antibodies included antibodies against CK19 (Dako, Clone RCK108), galectin-3 (Invitrogen, A3A12), HBME-1 (Dako, Clone HBME-1), and CD56 (Dako, Clone 123C3). The antigen retrieval buffer was EDTA (pH 9.0), the temperature was 98 °C, and the duration was 20 min. We used EnVision FLEX + Mouse LINKER to amplify the signal, the EnVision FLEX Mini Kit to visualize the immunohistochemistry (IHC) reaction, and the Autostainer Link 48 (Agilent Technologies, Santa Clara, CA, USA) to complete the procedure. The normal thyroid follicles around the nodules were the best IHC staining and evaluation controls for CD56. For CK19, galectin-3, and HBME-1, the known positive samples were put side by side with the target samples on each slide as controls.

Xiong Y., Li X., Liang L., Li D., Yan L., Li X., Di J, & Li T. (2021). Application of biomarkers in the diagnosis of uncertain samples of core needle biopsy of thyroid nodules. Virchows Archiv, 479(5), 961-974.

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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%).

Guo J., Hou L., Zhang W., Dong Z., Zhang L, & Wu C. (2021). Improving differential diagnosis of pulmonary large cell neuroendocrine carcinoma and small cell lung cancer via a transcriptomic, biological pathway-based machine learning model. Translational Oncology, 14(12), 101222.

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Immunohistochemical Analysis of NK Cells in Murine Tumor Models

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Tumors from the s.c and i.c mouse models were collected and fixed in 5% paraformaldehyde to confirm the presence of NK cells at tumor sites. They were then embedded in paraffin, sectioned into 4 μm coronal sections by using a microtome, and prepared for immunohistochemistry (IHC) staining. The antibodies used for the IHC experiment were anti-human CD56 (1:50 dilution; Code No. M7304; clone 123C3; DAKO; Denmark) and anti-human CD45RO (LCA; 1:100 dilution; Code No. M0701; clone 2B11+PD7/26; DAKO; Denmark). Tumor slides were scanned using the Aperio Scan Scope System (Aperio, Technology, Vista, CA, USA).

Tran T.A., Kim Y.H., Duong T.H., Thangaraj J., Chu T.H., Jung S., Kim I.Y., Moon K.S., Kim Y.J., Lee T.K., Lee C.W., Yun H., Lee J.J., Lee H.J., Lee K.H, & Jung T.Y. (2023). Natural killer cell therapy potentially enhances the antitumor effects of bevacizumab plus irinotecan in a glioblastoma mouse model. Frontiers in Immunology, 13, 1009484.

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Immunohistochemical Evaluation of Adenoma

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Following the diagnostic report, a representative slide was selected from each adenoma case. Four-micron paraffin sections were cut, dewaxed, hydrated, and submitted for immunohistochemistry. Immunohistochemical analysis with antibodies for Galectin3 (Cell Marque/SIGMA-ALDRICH, Monoclonal Mouse Antibody, Clone 9C4, Dilution 1:25), CK19 (Dako, Agilent, Monoclonal Mouse Antibody, Clone RCK108, Prediluted), CD56 (Dako, Agilent, Monoclonal Mouse Antibody, Clone 123C3, Dilution 1:100), and HBME1 (Dako, Agilent, Monoclonal Mouse Antibody, Clone HBME-1, Dilution 1:50) was performed in each case using an automatized immunostainer (Omnis, Agilent).

Sodo A., Verri M., Palermo A., Naciu A.M., Sponziello M., Durante C., Di Gioacchino M., Paolucci A., di Masi A., Longo F., Crucitti P., Taffon C., Ricci M.A, & Crescenzi A. (2020). Raman Spectroscopy Discloses Altered Molecular Profile in Thyroid Adenomas. Diagnostics, 11(1), 43.

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Immunohistochemical Profiling of Tissue Samples

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Unstained 3-μm-thick slides of formalin-fixed, paraffin-embedded tissues were obtained and submitted for immunostaining with an automated stainer (Dako Omnis, Glostrup, Denmark). The following primary antibodies were used: CD56 (pre-diluted, clone 123C3, Dako, Glostrup, Denmark), Glial Fibrillary Acidic Protein (GFAP) (1:200, clone 6F2, Dako, Glostrup, Denmark), Olig2 (1:500, clone OLIG2, Sigma-Aldrich, Saint-Louis, USA), vimentin (1:800, clone V9, Dako, Glostrup, Denmark), neurofilament (1:100, clone NF70, Dako, Glostrup, Denmark), NeuN (1:1000, clone A60, Sigma-Aldrich, Saint-Louis, USA), synaptophysin (1:150, clone Synap, Dako, Glostrup, Denmark), EMA (1:200, clone GM008, Dako, Glostrup, Denmark), CK18 (1:200, clone 6F2, Dako, Glostrup, Denmark), smooth muscle actin (1:4000, clone 1A4, Dako, Glostrup, Denmark), NFκB (1:6000, clone D14E12, Cell Signaling Technology, Danvers, USA), L1CAM (1:500, clone UJ127.11, Sigma-Aldrich, Saint-Louis, USA), and Ki-67 (1:200, clone MIB-1, Dako, Glostrup, Denmark). Reticulin staining was performed using the Reticulin silver plating kit according to Gordon & Sweets (Merck Millipore, Guyancourt, France). External positive and negative controls were used for all antibodies and staining.

Tauziède-Espariat A., Siegfried A., Nicaise Y., Kergrohen T., Sievers P., Vasiljevic A., Roux A., Dezamis E., Benevello C., Machet M.C., Michalak S., Puiseux C., Llamas-Gutierrez F., Leblond P., Bourdeaut F., Grill J., Dufour C., Guerrini-Rousseau L., Abbou S., Dangouloff-Ros V., Boddaert N., Saffroy R., Hasty L., Wahler E., Pagès M., Andreiuolo F., Lechapt E., Chrétien F., Blauwblomme T., Beccaria K., Pallud J., Puget S., Uro-Coste E, & Varlet P. (2021). Supratentorial non-RELA, ZFTA-fused ependymomas: a comprehensive phenotype genotype correlation highlighting the number of zinc fingers in ZFTA-NCOA1/2 fusions. Acta Neuropathologica Communications, 9, 135.

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Immunohistochemical Analysis of FFPE Tissues

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Formalin-fixed, paraffin-embedded tissue blocks were cut into 2 µm sections and transferred to glass slides (X-tra® adhesive, Leica Biosystems, Nussloch, Germany). After drying overnight at 37 °C, slides were deparaffinized with xylene and rehydrated with ethanol. For antigen retrieval slides were either subjected to water bath for 30 min at 92 °C according to manufacturer’s instructions (Trilogy-solution 1:100; Cell Marque Corporation, Rocklin, USA) followed by washing with water and TBS buffer. Following a 3 min wash with water, the slides were then processed on the Autostainer Link 48 (Dako, Glostrup, Denmark) using an automated staining protocol (Dako EnVision™ Flex, Code K8000). All primary antibodies were monoclonal mouse antibodies (Ki-67: clone MIB-1, 1:200, Dako; Chromogranin A: clone DAK-A3, 1:800, Dako; Synaptophysin: clone DAK-SYNAP, ready-to-use; CD56: Clone 123C3, ready-to-use) and exposition time was 30 minutes. Counterstaining was performed with hematoxylin.

Walter D., Harter P.N., Battke F., Winkelmann R., Schneider M., Holzer K., Koch C., Bojunga J., Zeuzem S., Hansmann M.L., Peveling-Oberhag J, & Waidmann O. (2018). Genetic heterogeneity of primary lesion and metastasis in small intestine neuroendocrine tumors. Scientific Reports, 8, 3811.

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Comprehensive Immune Profiling of Melanoma

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Dual IHC for MHC class I (HLA-A, HLA-B, and HLA-C, clone EMR8-5, 1:6000; Abcam) or MHC class II (HLA-DP, HLA-DQ, HLA-DR, clone CR3/43, 1:750; Dako) with the melanoma marker SOX10 (EP268, 1:1500; Cell Marque) was performed using an automated staining system (Bond-III; Leica Biosystems), as previously described (42) .
IHC for CD3 (LN10; Leica), CD4 (4B12; Dako), CD8 (C8/144B; Dako), PD-1 (NAT105; Abcam), CD56 (clone 123C3; Dako), TCR (clone H-41; Santa Cruz Biotechnology), and 2M (A0072, 1:6000; Dako) was performed either manually (CD3, CD4, CD8, and PD-1) or on Bond RX (2M, CD56, and TCR) per standard protocols. IHC for PD-L1 (28-8; Dako) was performed as part of an investigational use-only kit (PD-L1 IHC pharmDx) on Dako Link 48 (2) .

Rodig S.J., Gusenleitner D., Jackson D.G., Gjini E., Giobbie-Hurder A., Jin C., Chang H., Lovitch S.B., Horak C., Weber J.S., Weirather J.L., Wolchok J.D., Postow M.A., Pavlick A.C., Chesney J, & Hodi F.S. (2018). MHC proteins confer differential sensitivity to CTLA-4 and PD-1 blockade in untreated metastatic melanoma. Science translational medicine, 10(450).

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