Cd3 polyclonal

Manufactured by Agilent Technologies

Sourced in United States

The CD3 polyclonal is a laboratory reagent used for the detection and analysis of CD3-positive cells. It is a mixture of antibodies that bind to the CD3 protein, which is a component of the T-cell receptor complex. This product can be used in various immunological techniques, such as flow cytometry and immunohistochemistry, to identify and study T-cell populations.

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9 protocols using cd3 polyclonal

Immune Profiling of Tumor Tissue

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Tumor tissue was fixed in formalin and embedded in paraffin. For immunohistochemical staining, tissue was cut and mounted at a thickness of 4 μm per slide. Slides were then stained with CD3 polyclonal (1:100, DAKO), CD4 clone 4B12 (1:80, Leica Biosystems), CD8 clone C8/144B (1:25, Thermo Scientific), PD-L1 clone E1L3N (1:100, Cell Signaling Technology), PD-1 clone EPR4877-2 (1:250, Abcam), CD45RO clone UCHL1 (ready-to- use, Leica Biosystems), FoxP3 clone 206D (1:50, BioLegend), and Granzyme B clone F1 (ready-to-use, Leica Biosystems) [36 (link)] antibodies. Slides were then stained using diaminobenzidine as chromogen and the Leica Bond Polymer refine detection kit (Leica Biosystems). Slides were then counterstained with hematoxylin and scanned using an Aperio AT2 automated slide scanner (Leica Biosystems). Quantification was performed on 5 × 1 mm2 regions per tumor sample within the tumor center and measuring the average density of positive cells per region as a count of positive cells/mm2. For PD-L1, tumor proportion score was calculated by manual quantification with percentage between 0 and 100 % [37 (link)].

Akhave N., Zhang J., Bayley E., Frank M., Chiou S.H., Behrens C., Chen R., Hu X., Parra E.R., Lee W.C., Swisher S., Solis L., Weissferdt A., Moran C., Kalhor N., Zhang J., Scheet P., Vaporciyan A.A., Sepesi B., Gibbons D.L., Heymach J.V., Lee J.J., Wistuba I.I., Futreal P.A., Zhang J., Fujimoto J, & Reuben A. (2022). Immunogenomic profiling of lung adenocarcinoma reveals poorly differentiated tumors are associated with an immunogenic tumor microenvironment. Lung cancer (Amsterdam, Netherlands), 172, 19-28.

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Quantitative Immunohistochemistry of Tumor Immune Markers

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Tumor tissue was fixed in formalin and embedded in paraffin. For immunohistochemical staining, tissue was cut and mounted at a thickness of 4μm per slide. Slides were then stained with CD3 polyclonal (1:100, DAKO), CD4 clone 4B12 (1:80, Leica Biosystems), CD8 clone C8/144B (1:25, Thermo Scientific), PD-L1 clone E1L3N (1:100, Cell Signaling Technology), PD-1 clone EPR4877-2 (1:250, Abcam), CD45RO clone UCHL1 (ready-to-use, Leica Biosystems), FoxP3 clone 206D (1:50, BioLegend), and Granzyme B clone F1 (ready-to-use, Leica Biosystems)^{18 (link)}. Slides were then stained using diaminobenzidine as chromogen and the Leica Bond Polymer refine detection kit (Leica Biosystems). Slides were then counterstained with hematoxylin and scanned using an Aperio AT2 automated slide scanner (Leica Biosystems). Quantification was performed on 5 × 1 mm² regions per tumor sample within the tumor center and measuring the average density of positive cells per region as a count of positive cells/mm². For PD-L1, H-score was calculated by multiplying the proportion of positive cells in the sample (0–100%) by the intensity of staining (1⁺, 2⁺, or 3⁺) to obtain a score ranging between 1 and 300.

Reuben A., Zhang J., Chiou S.H., Gittelman R.M., Li J., Lee W.C., Fujimoto J., Behrens C., Liu X., Wang F., Quek K., Wang C., Kheradmand F., Chen R., Chow C.W., Lin H., Bernatchez C., Jalali A., Hu X., Wu C.J., Eterovic A.K., Parra E.R., Yusko E., Emerson R., Benzeno S., Vignali M., Wu X., Ye Y., Little L.D., Gumbs C., Mao X., Song X., Tippen S., Thornton R.L., Cascone T., Snyder A., Wargo J.A., Herbst R., Swisher S., Kadara H., Moran C., Kalhor N., Zhang J., Scheet P., Vaporciyan A.A., Sepesi B., Gibbons D.L., Robins H., Hwu P., Heymach J.V., Sharma P., Allison J.P., Baladandayuthapani V., Lee J.J., Davis M.M., Wistuba I.I., Futreal P.A, & Zhang J. (2020). Comprehensive T cell repertoire characterization of non-small cell lung cancer. Nature Communications, 11, 603.

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Immunocytochemical Analysis of Malignant Cells

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Immunocytochemical analysis was possible only on CBs, on three micron-thick unstained sections. An initial panel of antibodies was used in malignant cases to differentiate lymphoma from carcinoma and sarcoma, namely pan-cytokeratin (clone AE1/3; DAKO, Carpinteria, CA, USA), CD45/LCA (Clones 2B11+PD7/26; DAKO), CD20 (clone L26; DAKO), CD79α (clone JCB 117; DAKO), CD3 (polyclonal; DAKO), EMA (clone E29; DAKO), CD30 (clone Ber-H2; DAKO), CD15 (clone Carb-3; DAKO) and PAX5 (clone DAK-PAX5; DAKO). In case a diagnosis of B-lineage NHL had been advanced, a second lineage of immunocytochemical markers was performed, including BCL2 (clone 124; DAKO), BCL6 (clone PG-B6p; DAKO), CD10 (clone 56C6; DAKO), CD5 (clone 4C7; DAKO), CyclinD1 (clone EP12; DAKO), MUM1/IRF4 (clone MUM1p; DAKO) and Mib1 (clone Ki67; DAKO). All staining procedures were carried out on Autostainer Link48 (DAKO, DAKO), according to the manufacturer’s instructions and in the presence of appropriate controls. Neither morphology nor first- nor second-line immunocytochemical analyses aroused suspicion of T-lineage NHL.

Parente P., Covelli C., Zanelli M., Trombetta D., Carosi I., Carbonelli C., Sperandeo M., Mastracci L., Biancofiore G., Zizzo M., Taurchini M., Ascani S, & Graziano P. (2020). Diagnosis of Hodgkin Lymphoma from Cell Block: A Reliable and Helpful Tool in “Selected” Diagnostic Practice. Diagnostics, 10(10), 748.

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PD-L1 and CD3 Dual Immunohistochemistry

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All slides were baked for 60 min in an oven set to 60°C. They were then loaded into the Bond III staining platform with appropriate labels. Antigen retrieval was performed using Bond Epitope Retrieval 2 (Leica Biosystems) for 30 min. They were then incubated with PD-L1 (1:200; E1L3N, Cell Signaling Technology) for two applications of 60 min at RT. Primary antibody was detected using the Bond Polymer Refine Detection Kit (Leica Biosystems) and detected using DAB (3,3′-diaminobenzidine). Slides were then incubated in the second primary antibody CD3 (polyclonal) (Dako) at 1:250 for 30 min. Secondary antibody was detected using the Bond Polymer Refine Red Detection Kit (Leica Biosystems). Secondary antibody was developed using Fast Red, part of the detection kit. Slides were then dehydrated and coverslipped.

Ricklefs F.L., Alayo Q., Krenzlin H., Mahmoud A.B., Speranza M.C., Nakashima H., Hayes J.L., Lee K., Balaj L., Passaro C., Rooj A.K., Krasemann S., Carter B.S., Chen C.C., Steed T., Treiber J., Rodig S., Yang K., Nakano I., Lee H., Weissleder R., Breakefield X.O., Godlewski J., Westphal M., Lamszus K., Freeman G.J., Bronisz A., Lawler S.E, & Chiocca E.A. (2018). Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles. Science Advances, 4(3), eaar2766.

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Immunohistochemical Evaluation of B-Cell Lymphoma

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The B‐cell lymphoma diagnosis was made according to the 2017 WHO classification. Two hematopathologists reviewed the BM slides for the histopathological detection of BMI. Immunohistochemical staining was performed using monoclonal antibodies against the following antigens: CD3 (polyclonal, 1:200; Dako), CD5 (SP19, RTU; Roche), CD10 (56C6, 1:200; Novocastra), CD20 (L26, 1:4; Novocastra), CD79a (JCB117, 1:200; Dako), BCL2 (124, 1:200; Dako), BCL6 (GI191E/A8, RTU; Roche), cyclin D1 (SP4‐R, RTU; Roche), FMC7 (4C7, 1:100; Novocastra), Ki‐67 (MIB‐1, 1:100; Dako), and MUM‐1 (MUM1p, 1:400; Dako).

Jeon M.J., Yu E.S., Kim D.S., Choi C.W., Kim H.N., Kwon J.A., Yoon S, & Yoon J. (2024). Assessment of Bone Marrow Involvement in B‐Cell non‐Hodgkin Lymphoma Using Immunoglobulin Gene Rearrangement Analysis with Next‐Generation Sequencing. Journal of Clinical Laboratory Analysis, 38(6), e25027.

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Immunohistochemical Analysis of Bone Marrow

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Immunohistochemical studies were carried out in 38 of 45 cases using sections of decalcified paraffin-embedded bone marrow biopsy specimens. The following antibodies were used at the dilutions suggested by the manufacturers: CD3 (polyclonal, Dako), CD4 (clone 4B12, Dako), CD8 (clone C8/144B, Dako), CD20 (clone L26, Dako), CD56 (clone 123C3, Dako), CD57 (clone TB01, Dako), granzyme B (clone GrB-7, Dako), and T-cell restricted intracellular antigen 1 (TIA-1) (clone 2G9, Immunotech, France). After dewaxing and heat-induced antigen retrieval, immunostaining was performed on an Autostainer Link 48 (Dako, Denmark) according to the manufacturer’s instructions. All immunostained samples were counterstained with hematoxylin.

Gorodetskiy V.R., Sidorova Y.V., Kupryshina N.A., Vasilyev V.I., Probatova N.A., Ryzhikova N.V, & Sudarikov A.B. (2020). Analysis of a single-institution cohort of patients with Felty's syndrome and T-cell large granular lymphocytic leukemia in the setting of rheumatoid arthritis. Rheumatology International, 41(1), 147-156.

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Immunohistochemical Analysis of Tumor Markers

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Immunohistochemistry was performed on 2‐μm FFPE sections in an automated immunostainer (Autostainer Link48; Dako, Glostrup, Denmark) according to the manufacturer's instructions. The following antibodies were used: β‐catenin 14 (1 : 1000; BD Transduction, South San Francisco, CA, USA), CD3 polyclonal (1 : 400; Dako), CD4 4B12 (1 : 300; Dako), CD8 C8/144B (1 : 20; Dako), CD20 L26 (1 : 400; Dako), CD21 1F8 (1 : 50; Dako), CD56 123C3 (1 : 400; Dako), CD68 KP1 (1 : 3000; Dako), CD163 10D6 (1 : 200; Novocastra, Newcastle, UK), FOXP3 259D/C7 (1 : 200; BD Pharmingen), PD‐L1 SP142 (1 : 30; SPRING, Pleasanton, CA, USA), PD1 NAT105 (1 : 100; Biocare Medical, Pikenine, Concord, NC, USA), and EZH2 5246S (1 : 50; Cell Signaling, Leiden, Netherlands).

Colombo C., Belfiore A., Paielli N., De Cecco L., Canevari S., Laurini E., Fermeglia M., Pricl S., Verderio P., Bottelli S., Fiore M., Stacchiotti S., Palassini E., Gronchi A., Pilotti S, & Perrone F. (2017). β‐Catenin in desmoid‐type fibromatosis: deep insights into the role of T41A and S45F mutations on protein structure and gene expression. Molecular Oncology, 11(11), 1495-1507.

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Immunohistochemical Analysis of Spleen and Bone Marrow

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Immunohistochemical studies of the spleen (5 patients) and bone marrow (15 patients) were conducted using the formalin-fixed paraffin-embedded tissue. The following antibodies were used at dilutions suggested by the manufacturers: CD3 (polyclonal, Dako, Carpinteria, CA, United States); CD4 (clone 4B12, Dako); CD8 (clone C8/144B, Dako); CD16 (clone 2H7, Novocastra Laboratories, Newcastle upon Tyne, United Kingdom); CD20 (clone L26, Dako); granzyme B (clone GrB-7, Dako); T-cell restricted intracellular antigen 1 (TIA-1) (clone 2G9, Immunotech, France); TCR-β F1 (clone 8A3, Thermo Scientific, Waltham, MA, United States); and TCR-γ (clone γ3.20, Thermo Scientific). After dewaxing and heat-induced antigen retrieval, immunostaining was performed using an Autostainer Link 48 (Dako, Denmark) according to the manufacturer’s instructions. All immunostained samples were counter-stained with hematoxylin.

Gorodetskiy V., Sidorova Y., Biderman B., Kupryshina N., Ryzhikova N, & Sudarikov A. (2022). STAT3 mutations in “gray-zone” cases of T-cell large granular lymphocytic leukemia associated with autoimmune rheumatic diseases. Frontiers in Medicine, 9, 1000265.

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Immunohistochemical Analysis of CD3+ TILs

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Cut tissue sections (5 μm) on charged glass slides were baked for 10-12 hours at 58°C in a dry slide incubator, deparaffinized in xylene and rehydrated via an ethanol step gradient. Heat-induced antigen retrieval steps were performed at pH 9.0, with the primary antibody incubated at room temperature for 1 hour (CD3, polyclonal, Dako, 1:100) followed by standard chromogenic staining protocol with the Envision Polymer-HRP/3,3’diaminobenzidine (DAB, Dako) process. Slides were counterstained in Harris hematoxylin. Immunohistochemistry scoring was performed using the percentage of stromal CD3-positive tumor infiltrating lymphocytes (TILS). All IHC results were evaluated against positive and negative tissue controls.

Huang C., Chen L., Savage S.R., Eguez R.V., Dou Y., Li Y., da Veiga Leprevost F., Jaehnig E.J., Lei J.T., Wen B., Schnaubelt M., Krug K., Song X., Cieślik M., Chang H.Y., Wyczalkowski M.A., Li K., Colaprico A., Li Q.K., Clark D.J., Hu Y., Cao L., Pan J., Wang Y., Cho K.C., Shi Z., Liao Y., Jiang W., Anurag M., Ji J., Yoo S., Zhou D.C., Liang W.W., Wendl M., Vats P., Carr S.A., Mani D.R., Zhang Z., Qian J., Chen X.S., Pico A.R., Wang P., Chinnaiyan A.M., Ketchum K.A., Kinsinger C.R., Robles A.I., An E., Hiltke T., Mesri M., Thiagarajan M., Weaver A.M., Sikora A.G., Lubiński J., Wierzbicka M., Wiznerowicz M., Satpathy S., Gillette M.A., Miles G., Ellis M.J., Omenn G.S., Rodriguez H., Boja E.S., Dhanasekaran S.M., Ding L., Nesvizhskii A.I., El-Naggar A.K., Chan D.W., Zhang H, & Zhang B. (2021). Proteogenomic insights into the biology and treatment of HPV-negative head and neck squamous cell carcinoma. Cancer cell, 39(3), 361-379.e16.

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