Ab134047

Manufactured by Abcam

Sourced in United States, United Kingdom, China

Ab134047 is a primary antibody product for use in research applications. It is an immunoglobulin G (IgG) antibody that targets a specific protein or antigen. The core function of this product is to serve as a detection tool for the target molecule.

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Lab products found in correlation

102 protocols using ab134047

Immunostaining of Lung Tissue

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Mice were sacrificed with CO₂ exposure, and prior to harvest, the lungs were inflated with 4% PFA at a constant pressure of 25 cm H₂O. The lungs were fixed in 4% PFA overnight at 4°. The lung tissue was then dehydrated by immersing it in a series of ethanol solutions of increasing concentrations and embedded in paraffin for sectioning. Six-micrometer sections were immunostained with antibodies for VEGFR3 (R&D Systems, AF743) and VCAM (Abcam, ab134047). Human lung tissue was stained with antibodies for podoplanin (D240, BioLegend, 75782-960) and VCAM (Abcam, ab134047) overnight at 4°. After washing, the slides were incubated with Alexa Fluor-conjugated secondary antibodies for 1 h at room temperature. Slides were treated with DAPI-containing Vectashield that was used on the tissue sections, and a cover slip was applied. Secondary antibodies alone were used on the negative control slides to check for autofluorescence of lung tissue. Immunofluorescence was performed either using a Nikon Eclipse microscope and NIS Elements software or a Zeiss SP8 confocal microscope and Leica software. Image analysis was performed using ImageJ.

Trivedi A., Lu T.M., Summers B., Kim K., Rhee A.J., Houghton S., Byers D.E., Lis R, & Reed H.O. (2024). Lung lymphatic endothelial cells undergo inflammatory and prothrombotic changes in a model of chronic obstructive pulmonary disease. Frontiers in Cell and Developmental Biology, 12, 1344070.

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Immunohistochemical Quantification of Key Vascular and Oxidative Markers

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Antibodies against alpha smooth muscle actin (SMA, rabbit IgG, ab5694), MMP8 (rabbit IgG, ab53017), CD68 (rabbit IgG, ab125212), VCAM1 (rabbit IgG, ab134047), 8-Hydroxy-2′-deoxyguanosine (8-OHdG, mouse IgG, ab48508), and VCAM1 (rabbit IgG, ab134047) were purchased from Abcam, Cambridge, UK. Antibody against Angiotensin I (mouse IgG, sc-74528) was from Insight Biotechnology, UK. Antibody against Angiotensin II (rabbit IgG, ABIN6993662) was from Antibodies-online, Germany. Antibody against Ly6G (Rat IgG, 127602) was from Biolegend, London, UK. Monoclonal anti-α smooth muscle actin (SMA) (Clone 1A4, mouse IgG, A5228) was from Merck, Dorset, UK. All secondary antibodies and other materials were from ThermoFisher Scientific, Cheshire, UK, unless specifically indicated.

Zhang C., Niu K., Ren M., Zhou X., Yang Z., Yang M., Wang X., Luo J., Shao Y., Zhang C., Chen D., Gao S., Ge S., Wu Q, & Xiao Q. (2022). Targeted Inhibition of Matrix Metalloproteinase-8 Prevents Aortic Dissection in a Murine Model. Cells, 11(20), 3218.

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Multicolor Immunofluorescence of FFPE Tissues

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Multicolor immunofluorescence analyses were performed using 3-μm-thick sections of formalin-fixed paraffin-embedded (FFPE) tissues. Briefly, slides were deparaffinized in xylene and hydrated in a series of decreasing graded ethanol series. After heat-induced antigen retrieval in citrate buffer (pH = 6), samples were permeabilized with 0.5% Triton X-100, blocked with 5% goat serum-phosphate-buffered saline (PBS), and sequentially co-stained with antibodies recognizing VCAM-1 (Abcam, ab134047), CD248 (Abcam, ab217535), 4-HNE (Abcam, ab46545), FAPα (Invitrogen, BMS168; Abcam, ab218164), Mfap4 (Thermo, PA5-24865), Sparcl1 (Santa Cruz, sc-514275), F4/80 (Cell Signaling Technology, #70076), rabbit IgG Isotype Control (Invitrogen, 31235), mouse IgG Isotype Control (Invitrogen, 14-4714-82), and DAPI. A TSA indirect kit (PerkinElmer) was used according to the manufacturer’s instructions. Vectra® Polaris™ Imaging System (Akoya Biosciences) was used to collect images, and image analysis was performed using HALO^™ Image Analysis Software.

Wu J., Feng Z., Chen L., Li Y., Bian H., Geng J., Zheng Z.H., Fu X., Pei Z., Qin Y., Yang L., Zhao Y., Wang K., Chen R., He Q., Nan G., Jiang X., Chen Z.N, & Zhu P. (2022). TNF antagonist sensitizes synovial fibroblasts to ferroptotic cell death in collagen-induced arthritis mouse models. Nature Communications, 13, 676.

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Western Blot Analysis of Aortic Proteins

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Protein levels of genes in the aorta were detected by Western blotting analysis. In brief, aortic tissue lysates were separated by 10% SDS-PAGE and transferred to 0.45 μm PVDF membranes (IPVH00010, Merck Millipore). After blocking in 5% skim milk (Cat#D8340, Solarbio; for non-phosphorylated protein) or 5% BSA (G5001-5G, Servicebio; for phosphorylated protein) for 2 h, the membranes were incubated overnight at 4°C with primary antibodies: p65NF-κB (1:1000, AF5006, Affinity Biosciences); p-p65NF-κB (1:1000, AB76302, Abcam); p38 mitogen-activated protein kinase (p38MAPK; 1:2500, ab170099, Abcam); p-p38MAPK (1:1000, AB195049, Abcam); VCAM-1 (1:5000, ab134047, Abcam); and β-actin (1:1000, GB15003, Servicebio). The membranes were then incubated with HRP-conjugated secondary antibody for 90 min at room temperature. Protein bands were visualized using ECL solution on Ultra Sensitive Multifunctional Imager (AI680RGB, GE, Japan) and analyzed using the ImageJ software.

Li K., Song X., Li H., Kuang X., Liu S., Liu R, & Li D. (2024). Mussel oil is superior to fish oil in preventing atherosclerosis of ApoE−/− mice. Frontiers in Nutrition, 11, 1326421.

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Western Blot Analysis of Adenosine Receptors

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Tissues were homogenized using TissueLyser II (Qiagen) according to manufacturer’ instructions. Proteins were isolated using RIPA buffer (Boston BioProdcuts, BP-115) with protease inhibitor and phosphatase inhibitors. Protein concentrations were determined using Pierce BCA assay (Thermo Scientific). Twenty μg protein were loaded per lane on a 4–20% Mini-PROTEAN TGX Gel (Bio-Rad, 456–1096). Separated proteins were transferred to PVDF membranes using the Transfer Turbo Blot system (Bio-Rad) and Trans-Blot Turbo RTA Transfer Kit (Bio-Rad, 170–4272). The membrane was blocked with 5% nonfat milk in TBST for 1 hr at room temperature. After blocking, the membrane was incubated overnight at 4°C with antibodies against ADORA1 (1:500, ab82477, abcam), ADORA2A (1:750, ab3461, abcam), ADORA2B (1:750, ab40002, abcam), ADORA3 (1:500, ab203298, abcam), VCAM-1 (1:1000, ab134047, abcam), ICAM-1 (1:3000, BBA3, R and D Systems), E-Selectin (1:1000, BBA16, R and D Systems), p-AKT (1:1000, 4060, Cell Signaling), T-AKT (1:1000, 2920, Cell Signaling), β-actin (1:4000, 4970, Cell Signaling). Quantification of protein bands were performed using a luminescent image analyzer (BioRad, Chemidoc).

Yang D., Haemmig S., Zhou H., Pérez-Cremades D., Sun X., Chen L., Li J., Haneo-Mejia J., Yang T., Hollan I, & Feinberg M.W. (2021). Methotrexate attenuates vascular inflammation through an adenosine-microRNA-dependent pathway. eLife, 10, e58064.

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Multiparameter Immunophenotyping of Cells

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CD4-VioGreen (130-106-655, Miltenyi Biotec), Anti-CCR10-PE (clone: REA326, 130-104-822, Miltenyi Biotec), CD183 (CXCR3)-PE-Vio770 (clone: REA326, 130-104-822, Miltenyi Biotec), CD194 (CCR4)-APC (clone: REA279, 130-103-813, Miltenyi Biotec), CD196 (CCR6)- PE-Vivo-615 (clone: REA190, 130-107-142, Miltenyi Biotec), Propidium iodide (130-093-233, Miltenyi Biotec), NESTIN rabbit-anti-mouse (ab7659, Abcam), SCA1 rat-anti-mouse (ab25195, Abcam), PDGFRα goat-anti-mouse (AF1062, R&D Systems), Alexa Flour 405 donkey-anti-Rabbit (ab175651, Abcam), Alexa Flour 488 donkey-anti-goat (A-11055, Thermo Fisher Scientific), Alexa Flour 594 donkey-anti-rat (A-21209, Thermo Fisher Scientific), PerCP-CyTM 5.5 CD45.2 conjugated mouse-anti-mouse (552950, BD Biosciences), ICAM1 rabbit-anti-human (ab53013, Abcam), VCAM1 rabbit-anti-human (ab134047, Abcam), IL-1β rabbit-anti-human (ab2105, Abcam), TNF-α rabbit-anti-human (ab9635, Abcam), actin rabbit-anti-human (A2066, Sigma-Aldrich), rabbit anti goat (81–1620, Invitrogen), goat anti rabbit (65–6120,Invitrogen), CD3 rabbit-anti mouse (AD452, Dako), and B220 rat-anti-mouse (MAB1217, R&D Systems).

Dorraji S.E., Hovd A.M., Kanapathippillai P., Bakland G., Eilertsen G.Ø., Figenschau S.L, & Fenton K.A. (2018). Mesenchymal stem cells and T cells in the formation of Tertiary Lymphoid Structures in Lupus Nephritis. Scientific Reports, 8, 7861.

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Protein Extraction and Western Blot Analysis

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Total protein was extracted from HUVECs and kidney tissues using radioimmunoprecipitation assay lysis buffer (P0013B; Beyotime Institute of Biotechnology, Shanghai, China) containing phosphatase inhibitor cocktail (P1045; Beyotime), and protein concentrations were determined using BCA Protein Assay Kit (P0010; Beyotime). Protein extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrophoretically transferred onto nitrocellulose membranes (Pall Corporation, Cortland, NY, USA) at 100 V for 90 min. After blocking with 5% nonfat milk, membranes were incubated with anti-PI3Kγ (5405, 1:1,000; Cell Signaling Technology), pAkt (4060, 1:2,000, Cell Signaling Technology), Akt (4691, 1:1,000; Cell Signaling Technology), VCAM-1 (ab134047, 1:2,000; Abcam), and ICAM-1 (ab179707, 1:1,000; Abcam). Target proteins were examined using an electrochemiluminescence system (Thermo Fisher Scientific) and visualized using X-ray films (Carestream, Xiamen, China). β-Actin was used as a control. The results were measured using ImageJ [26 (link)] (version 1.52; National Institutes of Health).

Li H., Xu J.X., Cheng T.C., Tian L.J., Lin J.F., Luo X., Bian Z.L, & Han X.D. (2022). Inhibition of Phosphoinositide 3-Kinase Gamma Protects Endothelial Cells via the Akt Signaling Pathway in Sepsis-Induced Acute Kidney Injury. Kidney & Blood Pressure Research, 47(10), 616-630.

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Therapeutic Inhibitors Targeting Inflammation

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KCa3.1 inhibitor TRAM-34, NF-κB inhibitor PDTC and STAT3 inhibitor AG490 were purchased from MCE (Shanghai, China). All these inhibitors dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). Antibodies against KCa3.1 (60276-1-Ig) and Interleukin (IL)-1β (16806-1-AP) were purchased from Proteintech (Wuhan, China). Antibodies for inducible nitric oxide synthase (iNOS) (ab178945), matrix metalloproteinase (MMP)-9 (ab228402), vascular cell adhesion molecule (VCAM)-1 (ab134047), STAT3 (ab68153), phospho (p)-STAT3 (ab76315), horseradish peroxidase (HRP)-conjugated IgG (ab205718, ab205719) were purchased from Abcam (Cambridge, MA, USA). Antibodies against β-tubulin (15115S), NF-κB p65 (8242T), p-NF-κB p65 (3033T), p38 MAPK (8690T) and p-p38 MAPK (4511T) were purchased from CST (Danvers, MA, USA). Antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (BL006B) and β-actin (BL005B) were purchased from Biosharp (Hefei, China). Primary antibody for Histone H3 (EM30605) was purchased from HUABIO (Hangzhou, China)

Zheng F., Tao Y., Liu J., Geng Z., Wang Y., Wang Y., Fu S., Wang W., Xie C., Zhang Y, & Gong F. (2021). KCa3.1 Inhibition of Macrophages Suppresses Inflammatory Response Leading to Endothelial Damage in a Cell Model of Kawasaki Disease. Journal of Inflammation Research, 14, 719-735.

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Immunohistochemical Analysis of Liver Inflammation

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The liver specimens were embedded in paraffin and sectioned into 4-μm thick tissue slices. All sections were first stained with hematoxylin and eosin (H&E) for morphological structure analysis. For immunohistochemical staining (IHC), after antigen retrieval and endogenous peroxidase blocking, the sections were incubated with primary antibodies against TLR4 (1/100, 19811-1-AP, Proteintech, Wuhan, China), p-NF-κB p65 (1/50, ab194726, Abcam), IL-6 (use concentration of 1 μg/ml, ab9324, Abcam), and VCAM-1 (1/500, ab134047, Abcam) at 4 °C overnight and then incubated with secondary antibodies at room temperature for 2 h. Freshly prepared 3,3′-diaminobenzidine solution (Boster) was used for coloration. Next, the sections were counterstained with hematoxylin. The brown color indicated positive staining.

Tang D., Wang R.Y., Sun K.W., Wu Y., Ding L, & Mo Y. (2022). Network pharmacology-based prediction of active compounds in the Wenyang Jiedu Huayu formula acting on acute-on-chronic liver failure with experimental support in vitro and in vivo. Frontiers in Pharmacology, 13, 1003479.

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Western Blot Analysis of HPMEC Lysates

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Whole cell lysate was prepared from cultured HPMECs using 1× sample buffer (0.5 M Tris-HCL pH 6.8, 10% (w/v) SDS, 6% β-mercaptoethanol, 10% glycerol, 100 mM dithiothreitol, 0.1% bromophenol blue). Homogenates were centrifuged at 12000 rpm at 4°C for 2 min and then sonicated for 10 s (30 amplitude). Homogenates were then spun down again and boiled for 3 min before storing at −20°C until gel loading. Following separation by SDS–PAGE, proteins were transferred to a nitrocellulose membrane and assessed by Western blotting with primary antibodies against Poldip2 (ab181841; Abcam), VCAM-1 (ab134047; Abcam), β-actin (A5441; Sigma), or GAPDH (mab374; Millipore). Blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies depending on the species of the primary antibody (anti-mouse [NA931; GE] and anti-rabbit [70745; Cell Signaling]) and assessed using enhanced chemiluminescence (ECL, GE). HRP-induced luminescence was detected with Amersham Hyperfilm ECL (GE). Detected bands were scanned and densitometry was performed using ImageJ.

Forrester S.J., Xu Q., Kikuchi D.S., Okwan-Duodu D., Campos A.C., Faidley E.A., Zhang G., Lassègue B., Sadikot R.T., Griendling K.K, & Hernandes M.S. (2019). Poldip2 deficiency protects against lung edema and vascular inflammation in a model of acute respiratory distress syndrome. Clinical science (London, England : 1979), 133(2), 321-334.

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