Ab16669

Manufactured by Abcam

Sourced in United Kingdom, United States, China

Ab16669 is a monoclonal antibody that targets the CD3 epsilon subunit of the T cell receptor. It is suitable for use in various immunological applications such as flow cytometry, immunohistochemistry, and western blotting.

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Lab products found in correlation

Ab955, by Abcam (14 mentions) Ab15580, by Abcam (11 mentions) Ab125212, by Abcam (10 mentions) Ab217344, by Abcam (9 mentions) Ab183685, by Abcam (8 mentions) Saponin, by Merck Group (6 mentions) Protein block solution, by Agilent Technologies (6 mentions) Mca48r, by Bio-Rad (6 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions) Ab133357, by Abcam (5 mentions) Ab64100, by Abcam (5 mentions) Ab28364, by Abcam (5 mentions) Ab133616, by Abcam (5 mentions) Ab7817, by Abcam (5 mentions) Epi fluorescence microscopy system, by Olympus (4 mentions) A7811, by Merck Group (4 mentions) Tunel solution, by Roche (4 mentions) Ab6640, by Abcam (4 mentions) Ab31630, by Abcam (4 mentions) Ab205921, by Abcam (4 mentions)

Close spelling variants of this product

Anti-CD3 ab16669 (3 citations) CD3 (ab16669) (2 citations) CD3ϵ (clone ab16669, (2 citations)

182 protocols using ab16669

Formalin-Fixed Paraffin-Embedded Tumor Tissue Preparation

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Tumor tissues were harvested and fixed in 10% buffered formalin, dehydrated, cleared and embedded in paraffin according to standard procedures^{19 (link)}. They were then sectioned at 3 µm, mounted onto polylysine-coated slides, and the sections were stained with H&E. Histopathological morphology was checked using a microscope. For immunofluorescence assays, the sections were deparaffinized in xylene and rehydrated in graded ethanol. Antigen retrieval was performed in citrate buffer (pH 6.0) by boiling for 30 min in a microwave and cooling to room temperature. Then, the sections were blocked by 3% bovine serum albumin (BSA) in PBS for 1 h and 0.3% hydrogen peroxide (H2O2) solution in PBS for 10 min. The sections were then stained with antibodies against mouse CD3 (1:500, ab16669; Abcam, Cambridge, UK) at 4°C overnight. After washing, the sections were incubated with secondary antibody, AlexaFluor 488 (goat anti-rabbit, 1:500, ab16669; Abcam) for 1 h, and were then washed and mounted in Fluoroshield with DAPI (F6057; Sigma-Aldrich). Images were acquired using a Nikon Eclipse E400 microscope (Nikon, Tokyo, Japan) with an Olympus DP73 camera (Olympus, Tokyo, Japan) and cellSence Entry software (Olympus).

Chen T., Liu K., Xu J., Zhan T., Liu M., Li L., Yang Z., Yuan S., Zou W., Lin G., Carson D.A., Wu C.C, & Wang X. (2020). Synthetic and immunological studies on the OCT4 immunodominant motif antigen-based anti-cancer vaccine. Cancer Biology & Medicine, 17(1), 132-141.

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Immunohistochemical and Immunofluorescent Analyses

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For immunohistochemistry staining, formalin-fixed and paraffin-embedded tissue sections were incubated with primary antibodies against MTCO1 (ab14705, Abcam), CD68 (ab955, Abcam), or CD3 (ab16669, Abcam) and then analyzed using streptavidin peroxidase detection system (Maixin) according to the manufacturer’s protocol. Diaminobenzidine (DAB) (Maixin) was used as a horseradish peroxidase (HRP)–specific substrate. For immunofluorescence staining, formaldehyde-fixed cells or kidney sections were performed with primary antibodies against RFP (ab62341, Abcam), CD63 (sc5275, Santa Cruz Biotechnology), IL-10 (ab9969, Abcam), KIM-1 (MA5-28211, Invitrogen), CD68 (ab955, Abcam), iNOS (ab15323, Abcam), CD206 (ab64693, Abcam), and CD3 (ab16669, Abcam), followed by incubation with secondary antibodies. Cell nuclei were stained with DAPI. Immunostained samples were visualized under a confocal microscope (FV1000, Olympus).

Tang T.T., Wang B., Wu M., Li Z.L., Feng Y., Cao J.Y., Yin D., Liu H., Tang R.N., Crowley S.D., Lv L.L, & Liu B.C. (2020). Extracellular vesicle–encapsulated IL-10 as novel nanotherapeutics against ischemic AKI. Science Advances, 6(33), eaaz0748.

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Histological Analysis of Rat Kidney Fibrosis

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Paraffin-embedded rat kidney sections (4 μm thickness) were prepared by a routine procedure [17 (link)]. Sections were stained with periodic acid-Schiff reagent by standard protocol. Kidney sections were also subjected to Masson’s Trichrome staining for assessing collagen deposition and fibrotic lesions. Semi-quantitative determination of renal fibrosis score was carried out by previously reported methods [32 (link)]. Briefly, Masson trichrome-stained kidney sections were graded for the presence of interstitial fibrosis according to the following scale: 0, no evidence of interstitial fibrosis; 1, <25% involvement; 2, 25to 50% involvement, and 3, >50% involvement. The scale for each rat was reported as the mean of 10 random high-power (X400) fields per section [32 (link)]. Immunohistochemical staining was performed using established protocol, as described previously [9 (link)]. Antibodies used were as follow: rabbit polyclonal anti-α-smooth muscle actin antibody (ab5694), rabbit monoclonal anti-β-catenin antibody (ab32572), rabbit monoclonal anti-CD3 antibody (ab16669; Abcam, Cambridge, MA), mouse monoclonal anti-CD68 antibody (MCA341GA; Serotec), goat polyclonal anti-AGT (sc-7419), goat polyclonal anti-renin (sc-27320, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-AT1 receptor (AB15552; Millipore, Billerica, MA).

Xiao L., Xu B., Zhou L., Tan R.J., Zhou D., Fu H., Li A., Hou F.F, & Liu Y. (2019). Wnt/β-catenin regulates blood pressure and kidney injury in rats. Biochimica et biophysica acta. Molecular basis of disease, 1865(6), 1313-1322.

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Immunohistochemical Analysis of Spinal Cord Tissue

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The paraffin-embedded sections (4 μm) of the spinal cords were mounted on glass slides. IHC analysis of the sections was performed using an automated slide preparation system (Benchmark XT; Ventana Medical Systems Inc., Tucson, AZ, USA). Deparaffinization, epitope retrieval, and immunostaining were performed using cell conditioning solutions and the BMK ultraVIEW diaminobenzidine detection system (Ventana Medical Systems Inc., Tucson, AZ), following the manufacturer’s instructions. The tissue sections were stained with anti-CD3 (#ab16669, Abcam), anti-CD68 (#ab125212, Abcam), and anti-PAX5 (#ab109443, Abcam) primary antibodies. The positive signals were amplified using ultraVIEW copper. The sections were counterstained with hematoxylin and bluing reagent. The number of CD3-, CD68-, and PAX5-immunoreactive cells in the white matter and pia mater of the spinal cord was examined using a light microscope and normalized to the total area of white matter and pia mater.

Bae D., Lee J.Y., Ha N., Park J., Baek J., Suh D., Lim H.S., Ko S.M., Kim T., Som Jeong D, & Son W.C. (2021). CKD-506: A novel HDAC6-selective inhibitor that exerts therapeutic effects in a rodent model of multiple sclerosis. Scientific Reports, 11, 14466.

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Immunohistochemical Analysis of Cardiac Tissue

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For immunohistochemistry staining, heart cryosections were fixed with 4% paraformaldehyde, permeabilized and blocked with Protein Block Solution (DAKO, Carpinteria, CA) containing 0.1% saponin (Sigma, St Louis, MO), and then incubated with the following antibodies overnight at 4 °C: mouse anti-alpha sarcomeric actin (1:100, a7811, Sigma), rabbit anti-CD45 (1:100, ab10559, Abcam, Cambridge, United Kingdom), mouse anti-Actin, α-Smooth Muscle antibody (1:100, A5228, Sigma), rabbit anti-Ki67 (1:100, ab15580, Abcam), rabbit anti-CD3 (1:100, ab16669, Abcam) and mouse anti-CD8 alpha (1:100, mca48r, abd Serotec, Raleigh, NC ). FITC- or Texas-Red secondary antibodies (1:100) were obtained from Abcam Company and used for the conjunction with these primary antibodies. For assessment of cell apoptosis, heart cryosections were incubated with TUNEL solution (Roche Diagnostics GmbH, Mannheim, Germany) and counter-stained with DAPI (Life Technology, NY, USA). For assessment of angiogram, heart cryosections were incubated with Lectin (FL-1171, Vector laboratories, Burlingame, CA, USA). Images were taken by an Olympus epi-fluorescence microscopy system.

Tang J., Shen D., Caranasos T.G., Wang Z., Vandergriff A.C., Allen T.A., Hensley M.T., Dinh P.U., Cores J., Li T.S., Zhang J., Kan Q, & Cheng K. (2017). Therapeutic microparticles functionalized with biomimetic cardiac stem cell membranes and secretome. Nature Communications, 8, 13724.

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Automated Tumor Immune Profiling

Cited 1 time

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First, slides were mounted with tumor sections obtained from paraffin-embedded tumor blocks; then, one slide for each patient was used for immunohistochemistry staining (IHC) with anti-CD3 (ab16669, 1:800; Abcam, Cambridge, UK). All slides were subsequently scanned, and the digital images were used for further analyses. The digital pathology techniques consisted of nucleus segmentation and tumor cell (TC) and immune cell (IC) population classification as described in our previous study.^{5 (link)} Briefly, following stain deconvolution of the digital images, nucleus segmentation was performed in the hematoxylin channel using a regional convolutional neural network (R-CNN). Next, we classified the cells into TCs or ICs using the Xception deep learning model, which was developed based on the manual annotation of each nucleus as belonging to a TC or IC by two pathologists and achieved good performance (Supplementary Fig. S1).

Wang Y.Q., Liu X., Xu C., Jiang W., Xu S.Y., Zhang Y., Liang Y.L., Li J.Y., Li Q., Chen Y.P., Zhao Y., Yun J.P., Liu N., Li Y.Q, & Ma J. (2021). Spatial heterogeneity of immune infiltration predicts the prognosis of nasopharyngeal carcinoma patients. Oncoimmunology, 10(1), 1976439.

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Immunofluorescence Staining of Paraffin-Embedded Tissues

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All tissues were fixed in 10% formalin, sectioned, and embedded in paraffin or underwent H&E staining (Mayo Clinic Histology Core Facility). Immunofluorescence staining was performed based upon established protocols [28 ]. Briefly, slides were deparaffinized in a series of washes of decreasing ethanol content. CD3, GFP, CD11b-stained slides underwent heat-mediated antigen retrieval using sodium citrate buffer, while VV-stained slides used Tris/EDTA buffer. Slides were then stained with anti-CD3 (ab16669, Abcam, Cambridge, MA), anti-VV (ab35219, Abcam, Cambridge, MA), anti-GFP (ab6556, Abcam, Cambridge, MA), or anti-CD11b (ab133357, Abcam, Cambridge, MA) antibodies, followed by secondary staining with an AF568-tagged goat anti-rabbit antibody (A11011, Invitrogen, Carlsbad, CA) and counterstaining with DAPI. Images were acquired with an LSM780 confocal microscope and Zen software (Carl Zeiss, Thornwood, NY). Quantification was performed using ImageJ for tumor area calculation and blinded manual counting of CD3+ cells.

Schuelke M.R., Wongthida P., Thompson J., Kottke T., Driscoll C.B., Huff A.L., Shim K.G., Coffey M., Pulido J., Evgin L, & Vile R.G. (2019). Diverse immunotherapies can effectively treat syngeneic brainstem tumors in the absence of overt toxicity. Journal for Immunotherapy of Cancer, 7, 188.

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Immunostaining of Murine Melanoma Tumors

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Deparaffinization and antigen retrieval were performed in murine melanoma subcutaneous tumors using Antigen Retrieval Buffer (Abcam ab52488 and ab93684) and stained as previously described.^{20 (link)} The following antibodies were used for immune staining as described previously:^{5 (link)} anti-CD3 (ab16669), anti-CD4 (ab183685), anti-CD8 (ab22378), anti-granzyme B (ab4059), anti-F4/80 (ab6640), anti-iNOS (ab3523), anti-Arg.1 (ab91279), anti-LDHA (ab52488), and Goat Anti-Rabbit Alexa 488 (ab150077), all antibodies were purchased from Abcam. Images were obtained by using an Axio Observer Light Microscope with the Apotome.2 (Zeiss). Metastatic melanoma lesions were gated by generating a region of interest, and threshold merged fluorescence limited to ROI and calculated using the ImageJ software.

de Azevedo R.A., Shoshan E., Whang S., Markel G., Jaiswal A.R., Liu A., Curran M.A., Travassos L.R, & Bar-Eli M. (2020). MIF inhibition as a strategy for overcoming resistance to immune checkpoint blockade therapy in melanoma. Oncoimmunology, 9(1), 1846915.

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Immunohistochemical Analysis of Tumor Samples

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Tumor tissue was fixed in 4% paraformaldehyde (4% PFA, Boston BioProducts) and stored in 70% ethanol until further processing. IHC was performed by the Research Pathology Core at City of Hope. Briefly, paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E, Sigma-Aldrich), mouse anti-hPSCA antibody (Abnova, H00008000-M03, clone: 5C2), anti-mouse CD3 (Abcam, ab16669, clone: SP7), anti-mouse GzmB (Abcam, ab4059, polyclonal), anti-mouse CD8a (Cell Signaling, 98941S, clone: D4W2Z), anti-mouse PD-L1 (Abcam, AB80276, clone: MIH6), and anti-mouse FOXP3 (Abcam, clone: EPR22102-37) according to the manufacturer’s protocol. RNA in situ hybridization of hPSCA was performed by RNAScope (ACD). Images were obtained using the Nanozoomer 2.0HT digital slide scanner and the associated NDP.view2 software (Hamamatsu).

Murad J.P., Tilakawardane D., Park A.K., Lopez L.S., Young C.A., Gibson J., Yamaguchi Y., Lee H.J., Kennewick K.T., Gittins B.J., Chang W.C., Tran C.P., Martinez C., Wu A.M., Reiter R.E., Dorff T.B., Forman S.J, & Priceman S.J. (2021). Pre-conditioning modifies the TME to enhance solid tumor CAR T cell efficacy and endogenous protective immunity. Molecular Therapy, 29(7), 2335-2349.

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Quantifying T Cell Infiltration in Tumor Biopsies

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Tumors and other organs were removed from the mice and washed with PBS. All tissues were fixed with 4% paraformaldehyde overnight, then dehydrated by graded ethanol, vitrified by dimethyl benzene and finally embedded in paraffin. Serial 5.0 μm tissue biopsies were cut and used for H&E staining and IHC staining. The T cell infiltration in the tumors were marked by anti-CD3 antibodies (ab16669, Abcam). Image-pro Plus software was used to quantitatively calculate the sum area and sum IOD of the CD3 staining in tumor tissues. The necrosis and inflammation were compared between the two groups in a blind manner.

Chen J., Hu S., Ji D., Gao Z., Wang H., Yang Y., Chen Y, & Gu J. (2020). Hemolysin BL from novel Bacillus toyonensis BV-17 induces antitumor activity both in vitro and in vivo. Gut Microbes, 12(1), 1782158.

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