Cd3 clone ln10

Manufactured by Leica

Sourced in United Kingdom, Germany

The CD3 (clone LN10) is a lab equipment product from Leica. It is a cell surface marker that is used to identify and analyze T cells. The core function of the CD3 (clone LN10) is to detect and quantify T cells in biological samples.

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Clone LN10 (9 citations) Anti-CD3 (clone LN10, (8 citations) CD3 (LN10, (2 citations)

13 protocols using cd3 clone ln10

Immunohistochemical Analysis of Lymph Node Samples

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Four μm tissue sections from formalin-fixed and paraffin-embedded lymph nodes from the patient (PT) and two age-matched children were used for Hematoxylin and Eosin staining (H&E) and immunohistochemical staining to CD3 (clone LN10, Leica Biosystems), CD20 (clone L26, Leica Biosystems), and CD303 (clone 124B3.13, Dendritics) antigens. Briefly, after appropriate antigen retrival, antibodies were revealed using Novolink Polymer (Leica Biosystems) followed by Diaminobenzidine (DAB) and counterstained with hematoxylin.

Dotta L., Vairo D., Giacomelli M., Moratto D., Tamassia N., Vermi W., Lonardi S., Casanova J.L., Bustamante J., Giliani S, & Badolato R. (2020). Transient Decrease of Circulating and Tissular Dendritic Cells in Patients With Mycobacterial Disease and With Partial Dominant IFNγR1 Deficiency. Frontiers in Immunology, 11, 1161.

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Immunophenotyping of Tissue Samples

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All histological sections were reviewed, and 4-µm sections were obtained from the most representative formalin-fixed paraffin-embedded (FFPE) tissue blocks. Immunohistochemical stains (IHC) were performed utilizing antibodies directed against CD3 (clone LN10, dilution 1/250, Leica Biosystems, UK), CD4 (clone SP35, Ready to Use Predilute Antibody, Ventana, USA), CD8 (clone C8, dilution 1/250, Dako, Denmark), CD20 (clone L26, dilution 1/300, Dako, Denmark), and CD68 (clone KP1, dilution 1/1500, Dako, Denmark) utilizing clinically validated protocols. Slides were scanned at ×40 magnification on the Aperio GT450 brightfield instrument (Leica Biosystems). The resolution of the images was 0.26 µm/pixel at ×40. The images were 24-bit contiguous standard pyramid tiled TIFFs compressed via JPEG with a quality setting of 91. A board-certified neuropathologist selected and annotated regions for analysis using Aperio ImageScope Software (Leica Biosystems). The annotated regions of each stained slide were analyzed using proprietary nuclear and cytoplasmic algorithms. Cells within each region of interest were graded based on intensity of staining (0, 1 + , 2+ or 3 + ). Cells with an intensity of 1+ or higher were considered positive for immunostaining markers. Immunohistochemistry (IHC) scores were expressed as a percentage of positive cells (0 to 100) within the region of interest.

Galanis E., Dooley K.E., Keith Anderson S., Kurokawa C.B., Carrero X.W., Uhm J.H., Federspiel M.J., Leontovich A.A., Aderca I., Viker K.B., Hammack J.E., Marks R.S., Robinson S.I., Johnson D.R., Kaufmann T.J., Buckner J.C., Lachance D.H., Burns T.C., Giannini C., Raghunathan A., Iankov I.D, & Parney I.F. (2024). Carcinoembryonic antigen-expressing oncolytic measles virus derivative in recurrent glioblastoma: a phase 1 trial. Nature Communications, 15, 493.

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Immunohistochemical Profiling of Hematologic Malignancies

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Formalin-fixed paraffin-embedded tissues were stained with hematoxylin and eosin at the initial diagnosis. Immunohistochemistry was performed using the following panel of monoclonal and polyclonal antibodies: CD20 (clone L26, DAKO, Glostrup, Denmark); CD79a (clone 1.10E + 04, Leica Biosystems, Wetzlar, Germany); PAX5 (clone R1, DAKO); CD10 (clone 56C6, Leica Biosystems); BCL6 (clone P1F6, DAKO); MUM1 (clone MUM1p, DAKO); Ki-67 (clone MIB-1, DAKO); Terminal deoxynucleotidyl transferase (TDT, clone SP150, DAKO); CD2 (clone AB75, DAKO); CD3 (clone LN10, Leica Biosystems); CD4 (clone SP35, DAKO); CD7 (DAKO); CD8 (clone SP16, DAKO); CD1a (clone SP157, DAKO); CD34 (clone QBEnd/10, DAKO); CD43 (clone DF-T1, DAKO); CD99 (clone HO36-1.1, DAKO); CD117 (clone C-KIT, DAKO); CD45 (clone PAN-LCAL, DAKO); CD33 (clone WM-54, DAKO); MPO (clone SP72, DAKO); CD15 (clone C3D-1, DAKO); CD30 (clone Ber-H2, DAKO); CD23 (clone SP23, DAKO); and Epithelial membrane antigen (EMA, clone GP1.4, Leica Biosystems).

Chen L., Wang M., Fan H., Hu F, & Liu T. (2017). Comparison of pediatric and adult lymphomas involving the mediastinum characterized by distinctive clinicopathological and radiological features. Scientific Reports, 7, 2577.

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Immunohistochemical Profiling of DLBCL

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Formalin-fixed, paraffin-embedded tissue samples were available for all the 36 cases and were stained with hematoxylin and eosin (HE) at initial diagnosis. Immunohistochemistry was performed using a panel of monoclonal and polyclonal antibodies, as follows: CD20 (clone L26, DAKO, Glostrup, Denmark); CD79a (clone 1.10E+04, Leica Biosystems, Wetzlar, Germany); PAX5 (clone R1, DAKO); CD10 (clone 56C6, Leica Biosystems); BCL6 (clone P1F6, DAKO); MUM1 (clone MUM1p, DAKO); Ki-67 (clone MIB-1, DAKO); BCL2 (clone 100/D5, DAKO); MYC (DAKO); EBV (DAKO); terminal deoxynucleotidyl transferase (TDT, clone SP150, DAKO); CD3 (clone LN10, Leica Biosystems); CD43 (clone DF-T1, DAKO). MYC was considered as positive when > 40% of tumor cells exhibited staining [20 ]. Positivity threshold was defined at 50% for BCL2 [20 ], and at 30% for CD10, BCL6, and MUM1 [4 (link)]. For sub-classification of DLBCLs, GC immunophenotype was evaluated by antibodies of C10, BCL6 and MUM1 [4 (link)].

Chen L., Al-Kzayer L.F., Liu Y, & Liu T. (2017). B-cell lymphomas involving Waldeyer's ring characterized by distinctive clinical and histopathological features: a comparison of pediatric to adult patients. Oncotarget, 8(7), 11544-11554.

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Immunohistochemical Profiling of FFPE Skin Biopsies

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Skin biopsy specimens were routinely fixed in a 10% neutral-buffered formalin solution, and embedded in paraffin. The 4.5-µm sections of FFPE blocks were stained by H&E. Immunohistochemical analysis using the biotin-free alkaline phosphatase method (Leica Biosystems, Newcastle, UK) was performed using the fully automated stainer BOND-III systems (Leica Biosystems) with the following primary antibodies: CD3 (clone LN10, Leica Biosystems), CD4 (clone 4B12, DakoCytomation, Glostrup, Denmark), CD5 (clone 4C7, Leica Biosystems), CD20 (clone LR26, Leica Biosystems), CD56 (clone NCAM, Leica Biosystems), CD68 (clone 514H12, Leica Biosystems), CD123 (clone BR4MS, Novocastra, Leica Biosystems), TCL-1 (clone MRQ-7, Cell Marque, CA Rocklin, USA), TIA-1 (Biocare Medical, CA Pacheo, USA), and MPO (Cell Marque). Irrelevant IgG subclass‒matched antibodies were used for negative controls.

Shumilov E., Mazzeo P., Ghandili S., Künstner A., Weidemann S., Banz Y., Ströbel P., Pollak M., Kolloch L., Beltraminelli H., Kerkhoff A., Mikesch J.H., Schliemann C., Haase D., Wulf G., Legros M., Lenz G., Feldmeyer L., Pabst T., Witte H., Gebauer N, & Bacher U. (2024). Diagnostic management of blastic plasmacytoid dendritic cell neoplasm (BPDCN) in close interaction with therapeutic considerations. Annals of Hematology, 103(5), 1587-1599.

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Comprehensive Immunohistochemical Profiling of Tumor Microenvironment

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Immunohistochemistry (IHC) analysis was used to detect the MMR-related proteins MSH2, MSH6, MLH1, and PMS2. To assess the TIME, CD3, CD8, and PD-L1 expression were evaluated. IHC was performed using our laboratory protocol as described previously [22 (link), 23 (link)]. Briefly, 3-μm-thick TMA serial sections were deparaffinized and subjected to heat-induced epitope retrieval with 10 mM sodium citrate (pH 6.0) at 95°C for 20 min. Endogenous peroxidase activity was quenched using a 0.3% hydrogen peroxide solution.
TMA sections were incubated with primary antibodies against MLH1 (clone ES05, ready to use; Leica Biosystems), PMS2 (clone MOR4G, ready to use; Leica Biosystems), MSH2 (clone 25D12, ready to use; Leica Biosystems), MSH6 (clone PU29, ready to use; Leica Biosystems), CD3 (clone LN10, ready to use; Leica Biosystems), CD8 (clone 4B11, ready to use; Leica Biosystems), and PD-L1 (SP142, 1: 100, ZSGB-BIO, China). Human tonsils treated with primary antibodies were used as positive controls, while the same tissues without primary antibodies comprised the negative controls. After the reactions, all sections were counterstained with hematoxylin. All slides except those used for manual PD-L1 staining were stained using an automatic IHC staining instrument (BOND-III; Leica Biosystems, Wetzlar, Germany) according to the manufacturer's instructions.

Shi J., Yang X., Wang X., Luo Y., Zhou W., Luo H., Bianba Z., Nima Z., Wang Q., Wang H., Liao R., Ciren Q., Li M, & Pang J. (2022). Prevalence of Epstein–Barr Virus Infection and Mismatch Repair Protein Deficiency and the Correlation of Immune Markers in Tibetan Patients with Gastric Cancer. BioMed Research International, 2022, 2684065.

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Immunohistochemical Analysis of Gastric Cancer

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Serial 3-µm sections were prepared from one representative formalin-fixed paraffin-embedded (FFPE) block. IHC staining was performed in all GCLS cases with antibodies against cytokeratin (CK) AE1/AE3 (clone PCK26, prediluted; Ventana Medical Systems), CD3 (clone LN10, 1:500; Leica Biosystems), CD8 (clone SP57, prediluted; Ventana Medical Systems) and PD-L1 (clone E1L3N, 1:1000; Cell Signaling Technology) [17, (link)34, (link)35] (link). Samples were processed in the automatic Ventana Benchmark Ultra platform using the OptiView Universal DAB detection kit and the OptiView Amplification kit for PD-L1 staining. EBV infection was studied by chromogenic ISH for EBV-encoded RNA (EBER-ISH, INFORM EBER probe, Ventana Medical Systems) using the same equipment, with enzymatic digestion (ISH protease) and the iViewBlue detection kit. Two TMA 3 µm sections were prepared and used for PD-L1 IHC and EBER-ISH.

Gullo I., Oliveira P., Athelogou M., Gonçalves G., Pinto M.L., Carvalho J., Valente A., Pinheiro H., Andrade S., Almeida G.M., Huss R., Das K., Tan P., Machado J.C., Oliveira C, & Carneiro F. (2019). New insights into the inflamed tumor immune microenvironment of gastric cancer with lymphoid stroma: from morphology and digital analysis to gene expression. Gastric cancer : official journal of the International Gastric Cancer Association and the Japanese Gastric Cancer Association, 22(1).

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Immunohistochemical Staining Protocol

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Slides were run on the Bond III Imunostainer (deparaffinized on the machine) by Leica (Buffalo Grove, IL). The machine uses several retrievals including ER1 which is low pH (citrate based) and ER2 which is high pH (EDTA based) and heats the slides during the retrieval. The following antibodies were used: CD3 clone LN-10 (Leica), 1:300, 1 hour incubation with primary, retrieval ER2 20 minutes, detection with Bond Polymer Refine (DAB); CD8 clone C8/144R (Dako), 1:200, 1 hour incubation with primary, retrieval ER2 30 minutes, detection with Bond Polymer Refine; FOXP3/CD25 double stain: FOXP3 clone 206D (Biolegend), 1:50, retrieval ER2 40 minutes, detection with Bond Polymer Refine (DAB), CD25 clone 4C9 (Lifespan), 1:50, 1 hour incubation, detection with Bond Polymer Refine Red Detection.

van Galen P., Hovestadt V., Wadsworth M I.I., Hughes T., Griffin G.K., Battaglia S., Verga J.A., Stephansky J., Pastika T.J., Lombardi Story J., Pinkus G.S., Pozdnyakova O., Galinsky I., Stone R.M., Graubert T.A., Shalek A.K., Aster J.C., Lane A.A, & Bernstein B.E. (2019). Single-cell RNA-seq reveals AML hierarchies relevant to disease progression and immunity. Cell, 176(6), 1265-1281.e24.

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Dual Immunofluorescence for Transporter Localization

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Drug efflux transporter localization was identified using IHC as described previously (18 (link)). Dual immunofluorescence (IF) on frozen humanized mouse and NHP sections was performed on the Bond fully automated slide staining system (Leica Microsystems) using Bond Polymer Refine Detection kit (DS9800). Slides were allowed to sit at room temperature for 30 min and then fixed in 10% formalin for 15 min. They were then placed in Bond wash solution (AR9590). Antigen retrieval was done at 100°C in Bond epitope retrieval solution 2 (pH 9.0; AR9640) for 10 min. Staining was performed first using CD4 1F6 antibody (clone BC/1F6, Abcam) at 1:50 dilution for 1 hour with Bond polymer and post-primary reagents and Cy5 fluorochrome (PerkinElmer) for 15 min. Antigen retrieval was done at again 100°C in Bond epitope retrieval solution 2 (pH 9.0; AR9640) between protocols. Slides were then stained with CD3 (clone LN10, Leica) Ready-to-Use antibody for 15 min and Dako Envision mouse secondary for 30 min. Cy3 fluorochrome (PerkinElmer) was applied for 15 min. IF slides were counterstained with Hoechst 33258 (Invitrogen, Carlsbad, CA) and mounted with ProLong Gold antifade reagent (P36934, Life Technologies).

Thompson C.G., Rosen E.P., Prince H.M., White N., Sykes C., de la Cruz G., Mathews M., Deleage C., Estes J.D., Charlins P., Mulder L.R., Kovarova M., Adamson L., Arora S., Dellon E.S., Peery A.F., Shaheen N.J., Gay C., Muddiman D.C., Akkina R., Garcia J.V., Luciw P, & Kashuba A.D. (2019). Heterogeneous antiretroviral drug distribution and HIV/SHIV detection in the gut of three species. Science translational medicine, 11(499), eaap8758.

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Dual Immunofluorescence for Transporter Localization

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