Ab135372

Manufactured by Abcam

Sourced in United Kingdom

Ab135372 is a high-quality antibody product designed for use in various laboratory techniques. This product is intended for research purposes only.

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Lab products found in correlation

20 protocols using ab135372

Visualizing Blood Vessels and T Cells in Mouse Skin

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For visualization of blood vessels and CD3⁺ T cells in mouse ear skin, whole-mount immunofluorescence staining was carried out as previously described with minor modifications (52 (link)). In brief, mice were i.v. injected with 100 μL of TRITC-dextran (500 kDa, 10 mg/mL; MilliporeSigma, catalog 52194) 10 minutes before sacrifice. The dorsal side of the ear was carefully dissected, fixed in 4% paraformaldehyde, permeabilized in 0.3% Triton X-100 in PBS (PBST), and blocked with 3% skim milk in PBST. The ear skin was incubated with an anti-mouse CD3 antibody (Abcam, catalog ab135372, 1:200) at 4°C for 24 hours. After being washed in 0.05% Tween-20 in PBS, the samples were incubated with secondary antibodies at room temperature for 2 hours before washing and mounting. Images were captured by a confocal microscope (Carl Zeiss, LSM880). Negative controls that were stained with secondary antibodies were included in all experiments. Image analysis, including the calculation of blood vessel diameter and infiltrated T cells, was performed using ZEN software (Carl Zeiss, version 1.1.2.0) and ImageJ software. 3D reconstruction was performed using Imaris software (version 7.4.2).

Li Q., Shao S., Zhu Z., Chen J., Hao J., Bai Y., Li B., Dang E, & Wang G. (2023). An IGFBP7hi endothelial cell subset drives T cell extravasation in psoriasis via endothelial glycocalyx degradation. The Journal of Clinical Investigation, 133(9), e160451.

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Molecular Profiling of Therapeutic Targets

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Primary antibodies against caspase-3 (9665, CST), caspase-7 (ab32522, Abcam), CCT4 (ab205013, Abcam), GAPDH (97166, CST), PARP (9532, CST), p-STAT1 (Tyr701) (7649, CST), p-STAT2 (Tyr690) (4441, CST), p-STAT3 (Ser727) (9134, CST), p-STAT3 (Tyr705) (9145, CST), p-STAT5 (Tyr694) (4322, CST), p-STAT6 (Tyr641) (9361, CST), STAT1 (9172, CST), STAT3 (12640, CST), STAT5 (94205, CST), STAT6 (5397, CST), XIAP (2045, CST), LC3 (12741, CST), β-catenin (8480, CST), N-cadherin (13116, CST), β-actin (3700, CST), and secondary goat anti-mouse HRP-IgG (7076, CST) and goat anti-rabbit HRP-IgG (7074, CST) antibodies were used for immunoblotting. Primary antibodies against KI67 (9449, CST), CD3 (ab135372, Abcam), CD68 (76437, CST), CD56 (99746, CST), and secondary Fluorescein AffiniPure goat anti-rabbit IgG (111-095-144, Jackson) and Cy3 AffiniPure goat anti-rabbit IgG (111-165-144, Jackson) antibodies were used for immunofluorescence. Antibodies against p-STAT3 (Tyr705) (9145, CST), STAT3 (9139, CST), cleaved caspase-3 (Asp175) (9661, CST), and CCT4 (ab129072, Abcam) were used for immunohistochemistry.

Wang G., Zhang M., Meng P., Long C., Luo X., Yang X., Wang Y., Zhang Z., Mwangi J., Kamau P.M., Dai Z., Ke Z., Zhang Y., Chen W., Zhao X., Ge F., Lv Q., Rong M., Li D., Jin Y., Sheng X, & Lai R. (2022). Anticarin-β shows a promising anti-osteosarcoma effect by specifically inhibiting CCT4 to impair proteostasis. Acta Pharmaceutica Sinica. B, 12(5), 2268-2279.

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Comprehensive Histological Profiling of Immune Cells

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Histology, immunohistochemistry, and immunofluorescence were carried out as we described before.¹² Primary antibodies CD45 monoclonal antibody (ab208022, Abcam), F4/80 polyclonal antibody (ab100790, Abcam), CD3 monoclonal antibody (ab135372, Abcam), and MPO monoclonal antibody (ab208670, Abcam) were used for immunohistochemistry. Antibodies p‐Syk (ab62338, Abcam), p‐Syk (ab63515, Abcam), F4/80 polyclonal antibody (ab100790, Abcam), ZO‐1 (ab221547, Abcam), Occludin (ab216327, Abcam), and EpCAM (ab20160, Abcam) were employed for immunofluorescence. For mucus evaluation, slides were stained with Alcian Blue (AB; Sigma Aldrich) solution, pH 2.5, for 5 min and washed under running tap water.

Gong W., Yu J., Zheng T., Liu P., Zhao F., Liu J., Hong Z., Ren H., Gu G., Wang G., Wu X., Zhao Y, & Ren J. (2021). CCL4‐mediated targeting of spleen tyrosine kinase (Syk) inhibitor using nanoparticles alleviates inflammatory bowel disease. Clinical and Translational Medicine, 11(2), e339.

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Immunohistochemical Analysis of CD3 and CD31

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Paraffin-embedded sections first underwent standard deparaffinization and rehydration procedures and were then probed with fluorescein isothiocyanate-anti-CD3 antibody (ab135372; Abcam) and anti-CD31 antibody (ab9498; Abcam) overnight at 4°C [20 ]. Next, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies. The nuclei were then counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride [21 (link)].

Lu Y., Yang Y., Xiao L., Li S., Liao X, & Liu H. (2021). Autocrine and Paracrine Effects of Vascular Endothelial Cells Promote Cutaneous Wound Healing. BioMed Research International, 2021, 6695663.

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Antidifferentiation of T-cell Subsets

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Antidifferentiation cluster CD3, CD8, and interferon-γ (IFN-γ) antibodies were used to detect CD3⁺CD8⁺IFN-γ⁺ cells by flow cytometry. The cells were incubated for 30 min at 4°C with rat monoclonal antibodies: fluorescein isothiocyanate- (FITC-) conjugated antibodies (Abcam) to CD8 (ab22378, 1: 500) and IFN-γ (ab175878, 1: 100) and PE-conjugated antibody to CD3 (ab135372, 1: 200, Abcam). Following the fixing with 1% paraformaldehyde in PBS, cells were analyzed by a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Allotype-matched antibodies were adopted to control nonspecific staining subtracted from the specific staining results. At least 10,000 cells per sample were analyzed with the WinMDI 2.8 software (J. Trotter, Scripps Research Institute, La Jolla, CA).

Guo L.M., Ding G.F., Xu W.C., Ge H., Jiang Y, & Lu Y.F. (2022). Anti-PD-L1 Antibody Enhances T Cell Immune Responses and Reduces Resistance of Breast Cancer Cells to Radiotherapy. Oxidative Medicine and Cellular Longevity, 2022, 5938688.

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Immunohistochemical Detection of CD3 and Granzyme

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The tumor tissue was fixed, embedded in paraffin, and cut into 5‐μm‐thick sections, which were placed onto glass slides. The sections were then deparaffinized, rehydrated, and subjected to antigen retrieval. Next, the slides were blocked with goat serum and incubated with rabbit mAbs (1 : 200 dilution) against CD3 (ab135372; Abcam) or granzyme and then with a horseradish peroxidase‐labeled anti‐rabbit antibody (46890; Cell Signaling). Finally, the slides were developed with diaminobenzidine and counterstained in hematoxylin. Target protein expression was scored semiquantitatively according to a routine IHC grading system, and the values were used for statistical analysis.

Zhang H., Liu P., Zhang Y., Han L., Hu Z., Cai Z, & Cai J. (2021). Inhibition of galectin‐3 augments the antitumor efficacy of PD‐L1 blockade in non‐small‐cell lung cancer. FEBS Open Bio, 11(3), 911-920.

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FOXP3 and CD3 Expression via IHC

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The IHC assays were performed using an IHC kit (PV6000, ZSBIO, Beijing, China), and all the steps were performed according to the instructions. Importantly, the antibody (Anti-FOXP3, Abcam, ab215206, 1:500; Anti-CD3, Abcam, ab135372, 1:500) was incubated overnight. After staining, the IHC results were determined using ImageJ (v1.8.0_172) in Windows.

Guo L., Xie H., Zhang Z., Wang Z., Peng S., Niu Y, & Shang Z. (2021). Fusion Protein Vaccine Based on Ag85B and STEAP1 Induces a Protective Immune Response against Prostate Cancer. Vaccines, 9(7), 786.

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CD4+ T Cell Proliferation and Apoptosis Assays

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Based on previous work, we conducted cell proliferation and an apoptosis assay.^{29 (link)} CD4⁺T cells transfected with plasmids or treated with exosomes for 5 days were stained with 5 mM 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (MedChemExpress, Monmouth Junction, NJ, USA) to form fluorescent cells (1 × 10⁷ cells/ml). All cells were incubated with 1 mg/ml of anti-CD3 monoclonal antibodies (1:200, ab135372, Abcam) at 37°C and 1 mg/ml of anti-CD28 monoclonal antibodies (1:000, ab85986, Abcam) and coated in a 96-well plate at 4°C overnight for further use. Cell proliferation was analyzed using an Accuri C6 flow cytometer (Accuri Cytometers, Ann Arbor, MI, USA).
CD4⁺T cells (1 × 10⁴ cells/ml) were transfected with plasmids or treated with exosomes for 5 days after 18 h of incubation with 1ml of culture medium. CD4⁺T cells were then stained with Annexin V-FITC and 5 μg/ml of propidium iodide (Sigma-Aldrich, Madrid, Spain) in the dark for 15 min. All cells were collected and processed by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA) to analyze cell apoptosis.

Liu R., Jiang C., Li J., Li X., Zhao L., Yun H., Xu W., Fan W., Liu Q, & Dong H. (2021). Serum-derived exosomes containing NEAT1 promote the occurrence of rheumatoid arthritis through regulation of miR-144-3p/ROCK2 axis. Therapeutic Advances in Chronic Disease, 12, 2040622321991705.

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Immunohistochemical Staining of Paraffin Sections

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Paraffin embedded sections were deparaffinized and rehydrated at the Laboratory of Comparative Pathology at WCM. The sections were stained using the following antibodies: CD3 (Abcam ab135372, 1:250 following heat-induced epitope retrieval (HIER) in a pH 6.0 buffer); myeloperoxidase (Dako A0398, 1:1000, HIER pH 6.0); and F4/80 (Abcam, ab6640, 1:100, HIER in 10mM citrate buffer, pH6.0). IHC was performed on a Leica Bond RX automated stainer using Bond reagents (Leica Biosystems, Buffalo Grove, IL), including a polymer detection system (DS9800, Novocastra Bond Polymer Refine Detection, Leica Biosystems). The chromogen was 3,3 diaminobenzidine tetrachloride (DAB), and sections were counterstained with hematoxylin.

Sokhi U.K., Xia Y., Sosa B., Turajane K., Nishtala S.N., Pannellini T., Bostrom M.P., Carli A.V., Yang X, & Ivashkiv L.B. (2022). Immune Response to Persistent Staphyloccocus aureus Periprosthetic Joint Infection in a Mouse Tibial Implant Model. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 37(3), 577-594.

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Evaluating LY's Impact on Tumor Fibrosis and Lymphocyte Infiltration

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To evaluate whether LY inhibits tumor fibrosis and promotes lymphocyte infiltration in the tumor, 4T1 tumor-bearing mice were intratumor injected with LY (0.75 or 1.5 mg/kg) every other day. After one week, all mice were sacrificed and the tumors were sampled, Masson trichrome staining and immunohistochemical staining with anti-CD3 antibody (Abcam, ab135372, 1:150) were subsequently performed on the tumor sections (n = 3), finally measured by KF-PRO-120 digital pathology slide scanner (KFBIO Co., Ltd.). The depth of penetration into tumor sections of CD3⁺ T cells was estimated by ZEISS software. For evaluation of the CD3⁺ T cells (CD45⁺CD3⁺) and CD8⁺ T cells (CD45⁺CD3⁺CD8⁺), the T lymphocytes were stained with anti-CD45-APC (BD, 559864, 1:100), anti-CD3-PerCP-Cy5.5 (BD, 551163, 1:100) antibodies according to the manufacturer’s protocols, and further examined with flow cytometry.

Zhou M., Wang J., Pan J., Wang H., Huang L., Hou B., Lai Y., Wang F., Guan Q., Wang F., Xu Z, & Yu H. (2023). Nanovesicles loaded with a TGF-β receptor 1 inhibitor overcome immune resistance to potentiate cancer immunotherapy. Nature Communications, 14, 3593.

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