Cd3 clone sp7

Manufactured by Abcam

Sourced in United Kingdom, Japan

CD3 (clone SP7) is a monoclonal antibody that recognizes the CD3 complex, a key component of the T cell receptor (TCR) complex. The CD3 complex plays a crucial role in T cell activation and signal transduction. The SP7 clone is commonly used for the detection and quantification of CD3-positive T cells in various applications, such as flow cytometry and immunohistochemistry.

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Lab products found in correlation

Vector novared, by Vector Laboratories (2 mentions) Poly hrp anti rabbit igg, by Immunologic (2 mentions) Lsab system hrp, by Agilent Technologies (1 mentions) Bx51 fluorescent microscope, by Olympus (1 mentions) Tissue studio 3, by Definiens (1 mentions) Cd8 clone 144b, by Abcam (1 mentions) Horseradish peroxidase hrp labeled secondary antibody, by Agilent Technologies (1 mentions) 3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions) Opal ihc kit, by PerkinElmer (1 mentions) Vectra ver 3, by PerkinElmer (1 mentions)

5 protocols using cd3 clone sp7

CD3+ T Cell Quantification in Paraffin-Embedded MycCaP Tumors

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Paraffin-embedded MycCaP tumors were stained for CD3 expression by immunohistochemistry as described (18 (link)). Sections were stained with primary antibodies (CD3: clone SP7, Abcam), developed using the LSAB⁺ System-HRP (Agilent Technologies, Santa Clara, CA) and Metal Enhanced DAB Substrate Kit DAB metal concentration (Thermo Fisher Scientific, Waltham, MA), imaged using an Olympus BX51 fluorescent microscope (Olympus, Lombard, IL) in combination with SPOT RT analysis software (SPOT Imaging Solutions, Sterling Heights, MI), and quantified by the frequency of CD3⁺ cells per 10× field, counting at least five fields per tumor section per animal by a blinded investigator.

Olson B.M., Gamat M., Seliski J., Sawicki T., Jeffery J., Ellis L., Drake C.G., Weichert J, & McNeel D.G. (2017). Prostate cancer cells express more androgen receptor (AR) following androgen deprivation, improving recognition by AR-specific T cells. Cancer immunology research, 5(12), 1074-1085.

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Quantitative Analysis of Immune Cell Infiltration in CT26 Tumor Bearing Mouse Lungs

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Lungs of CT26 tumour bearing mice were fixed overnight in 4% phosphate buffered formaldehyde solution (Carl Roth) and embedded in paraffin. 50-μm consecutive sections (3 per mouse) were stained for CD3 (clone SP7, Abcam), CD4 (clone 1, catalogue no. 50134-M08H, Sino Biologinal) and FoxP3 (polyclonal, catalogue no. NB100-39002, Novus Biologicals) following detection by a HRP-conjugated antibody (Poly-HRP-anti-rabbit IgG, ImmunoLogic) and the corresponding peroxidase substrate (Vector Nova Red, Vector Laboratories) and counterstained with hematoxylin. CD3⁺, CD4⁺, FoxP3⁺ and tumour areas were captured on an Axio Scan.Z1 (Zeiss) and manually pre-defined tumour and lung regions were quantified via computerized image analysis software (Tissue Studio 3.6.1, Definiens). CD8⁺ area was calculated by subtracting CD4 stained area from CD3⁺ area. For comparison of tumour areas between control and pentatope1 +2-treated animals, tumour-free sections were excluded.

Kreiter S., Vormehr M., van de Roemer N., Diken M., Löwer M., Diekmann J., Boegel S., Schrörs B., Vascotto F., Castle J.C., Tadmor A.D., Schoenberger S.P., Huber C., Türeci Ö, & Sahin U. (2015). Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature, 520(7549), 692-696.

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Immunohistochemical Analysis of T-cell Subsets

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Standard immunohistochemical analysis was performed against primary antibody directed CD3 (clone SP7, diluted at 1: 100, Abcam, Cambridge, UK), CD4 (clone B468A1, diluted at 1: 200, Santa Cruz, Texas, USA) and CD8 (clone 144B, diluted at 1: 100, Abcam, Cambridge, UK). Antigen retrieval was achieved using EDTA buffer (pH 9.0) in a pressure cooker for 20 minutes. After neutralization of endogenous peroxidase, tissue microarray slides were pre-incubated with blocking serum and then were incubated with primary antibody for 45 minutes at room temperature. After three five-minute washes with PBS, the slides were treated with the horseradish peroxidase (HRP)-labeled secondary antibody (Dako, Glostrup, Denmark) for 20 minutes at room temperature, then washed in PBS. Finally, the reaction products were visualized with 3,3′-diaminobenzidine (DAB, Dako, Glostrup, Denmark) and the slides were counterstained with hematoxylin. After being dehydrated, slides were mounted in resin.

Chen K., Zhu Z., Zhang N., Cheng G., Zhang F., Jin J., Wu J., Ying L., Mao W, & Su D. (2017). Tumor-Infiltrating CD4+ Lymphocytes Predict a Favorable Survival in Patients with Operable Esophageal Squamous Cell Carcinoma. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 23, 4619-4632.

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Quantifying Tumor-Infiltrating Lymphocytes by IHC

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Explanted tumors were analyzed by IHC. Tumors were formalin-fixed paraffin embedded (FFPE). Sections were stained using standard immunohistochemistry (IHC) techniques. Areas of necrosis or hemorrhage, identified on corresponding H&E-stained sections, were excluded from TIL scoring and two blinded individuals scored each section. 50-µm consecutive sections (3 per mouse) were stained for CD3 (clone SP7, Abcam), GZMB (clone 1, catalogue no. 50134-M08H, Sino Biologinal) and FoxP3 (polyclonal, catalogue no.NB100-39002, Novus Biologicals) and detected by a secondary HRP-conjugated antibody (Poly-HRP-anti-rabbit IgG, ImmunoLogic). Reaction was visualized by incubation with the peroxidase substrate (Vector Nova Red, Vector Laboratories). Each sample was analyzed by counting cells in 10 independent areas.

Petrizzo A., Mauriello A., Luciano A., Rea D., Barbieri A., Arra C., Maiolino P., Tornesello M., Gigantino V., Botti G., Ciliberto G., Buonaguro F.M., Tagliamonte M, & Buonaguro L. (2017). Inhibition of tumor growth by cancer vaccine combined with metronomic chemotherapy and anti-PD-1 in a pre-clinical setting. Oncotarget, 9(3), 3576-3589.

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Multiplex Immunohistochemistry for Immune Cell Profiling

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For immune cell profiling we conducted mfIHC. Details of the procedure have been described previously [28 (link)]. Tyramide signal amplification was used employing an Opal IHC kit (PerkinElmer, Waltham, MA) according to the manufacturer's instructions. We previously constructed a T-cell panel, comprising CD3, CD4, CD8, FOXP3 and T-bet. This panel also included cytokeratin to differentiate tumor and stromal areas, and DAPI for staining nuclei [28 (link)]. Primary antibodies used were: CD3 (clone SP7, Abcam, Tokyo, Japan), CD4 (clone 4B12, Leica Microsystems, Tokyo, Japan), CD8 (clone 4B11, Leica Microsystems), FOXP3 (D6O8R, Cell Signaling Technology, Danvers, MA), T-bet (clone 4B10, Santa Cruz Biotechnology, Dallas, CA) and cytokeratin (clone AE1/AE3, Dako Agilent Technologies, Santa Clara, CA). Employing an automated imaging system (Vectra ver. 3.0, PerkinElmer), an average of 20 areas at × 200 were captured for each patient. An image analyzing software program (InForm, PerkinElmer) segmented cancer tissue into cancer cell nests (intratumoral) and the framework (stromal) region, and detected immune cells with specific phenotypes. Representative images of tissue segmentation and cell phenotype recognition are shown in Additional file 2. Infiltrating immune cells were quantified using an analytic software program (Spotfire, TIBCO, Palo Alto, CA) and then calculated per area.

Onagi H., Horimoto Y., Sakaguchi A., Ikarashi D., Yanagisawa N., Nakayama T., Nakatsura T., Ishizuka Y., Sasaki R., Watanabe J., Saito M., Saeki H., Hayashi T., Arakawa A., Yao T, & Kitano S. (2022). High platelet-to-lymphocyte ratios in triple-negative breast cancer associates with immunosuppressive status of TILs. Breast Cancer Research : BCR, 24, 67.

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