Hippocampa; proteins (30 μg) were separated by 12% SDS-PAGE, and the gel was cut according to the mass marker. Then the targeted proteins were transferred to 0.45 nm polyvinylidene difluoride (PVDF) membranes. After blocking in TBST buffer (1X TBS containing 0.1% Tween 20) with 5% (w/v) skimmed milk for 40 min at room temperature, the membranes were incubated with diluted primary antibodies (anti-VCAM-1 antibody; anti-S100A8 + S100A9 antibody; anti-CaMKK2 antibody; anti-MKK7 antibody; Abcam, China) at a dilution of 1:1000 with gentle shaking overnight at 4 °C. After washing 3 times with TBST buffer, the membrane was probed with secondary antibodies (dilution 1:5000) coupled to horseradish peroxidase at room temperature for 1 h. Densitometry analysis was performed using ImageJ software, and protein-to-β-actin ratios were expressed as the means ± SD from four independent experiments. P values were calculated using one-way ANOVA.
Anti vcam 1 antibody
Manufactured by Abcam
Sourced in United States
Anti-VCAM-1 antibody is a laboratory reagent used to detect and quantify the presence of the Vascular Cell Adhesion Molecule 1 (VCAM-1) in biological samples. VCAM-1 is a cell surface glycoprotein involved in cell-cell adhesion processes.
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Lab products found in correlation
Anti icam 1 antibody, by Abcam (3 mentions)
Anti icam 1, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti vinculin, by Santa Cruz Biotechnology (1 mentions)
Anti vimentin, by Santa Cruz Biotechnology (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Anti ilk, by Santa Cruz Biotechnology (1 mentions)
Anti cofilin 1, by Santa Cruz Biotechnology (1 mentions)
Coomassie staining, by Merck Group (1 mentions)
Anti nephrin antibody, by Abcam (1 mentions)
Anti tenascin c antibody, by Santa Cruz Biotechnology (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Rabbit anti human igg h l hrp, by Abcam (1 mentions)
Ripa buffer, by Abcam (1 mentions)
Bca kit, by Beyotime (1 mentions)
Anti il 1β antibody, by Abcam (1 mentions)
Cytotox onetm homogeneous membrane integrity assay, by Promega (1 mentions)
Dcfh da, by Thermo Fisher Scientific (1 mentions)
Anti p iκbα antibody, by Cell Signaling Technology (1 mentions)
Anti lamin b1 antibody, by Abcam (1 mentions)
9 protocols using anti vcam 1 antibody
1
Westerm Blot Analysis of Hippocampal Proteins
2
SPION Microbubble Functionalization for VCAM-1 Targeting
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SPION microbubbles were dispersed in double distilled water, which was put in a centrifuge at 1000 rpm for 3 minutes, and the supernatant was discarded. SPIONs were dispersed with 0.1 mol/L MES buffer (pH 6.0), which was placed in a centrifuge at 1000 rpm for 3 min, and the supernatant was discarded. SPIONs were dispersed again by 0.1 mol/L MES buffer (PH6.0). SPIONs were added with coupling agent EDC/NHS (PLGACOOH : EDC : NHS molar ratio 1 : 10 : 20) and incubated for 30 min. Rinse 2 times with MES buffer (pH 6.0), then rinse once with MES buffer (pH 8.0), centrifuge at 1000 rpm for 3 minutes, and discard the supernatant. The contrast agent was dispersed in MES buffer (pH 8.0) and added with excess anti-VCAM-1 antibody (Abcam, USA). The molar ratio of anti-VCAM-1 antibody : contrast agent was 50 : 1 and incubated for 2 h at room temperature. Rinse twice with PBS buffer to remove unbound anti-VCAM-1 antibody. Rinse twice with pH 7.0PBS buffer, centrifuge at 1000 rpm for 3 min, discard the supernatant to retain the lower layer, and add PBS to disperse.
3
Immunoblotting of Cell Protein Markers
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After electrophoresis of total cell proteins, samples were immunoblotted as previously reported82 (link),83 (link). Membranes were then incubated overnight at 4 °C with the following polyclonal antibodies [dilution, -fold]: anti-VCAM1 antibody (Abcam,) [5000], anti-E-Cadherin antibody (BS Transduction Laboratories) [1000], anti-tenascin-C antibody (Santa Cruz Biotechnology, Santa Cruz CA) [500], anti- NPHS2 antibody (Abcam) [5000], anti-nephrin antibody (Abcam) [5000] and anti-tubulin (Sigma–Aldrich, Saint Louis, MO [5000]. Anti-vimentin (Santa Cruz Biotechnology) [1000], anti-cofilin-1 (Santa Cruz Biotechnology) [1000], anti-vinculin (Santa Cruz Biotechnology) [1000], anti-ILK (Santa Cruz Biotechnology) [1000], anti-β-catenin (Santa Cruz Biotechnology) [2000].
Coomassie staining (Sigma) of the membrane was used as an internal loading control. Blots were analyzed by densitometric scanning with Image J. Western blot studies in cultured cells were performed in at least three independent experiments, and a representative figure is shown.
Coomassie staining (Sigma) of the membrane was used as an internal loading control. Blots were analyzed by densitometric scanning with Image J. Western blot studies in cultured cells were performed in at least three independent experiments, and a representative figure is shown.
4
Protein Extraction and Western Blot Analysis
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Total protein extracted from cells using RIPA buffer (Abcam) was quantified with a BCA Kit (Beyotime). Western blot analysis was carried out as previously reported.26 All antibodies used in this study were purchased from Abcam: Anti‐BMP4 antibody (ab235114), Anti‐ICAM1 antibody (ab171123), Anti‐VCAM1 antibody (ab134047), Anti‐VEGFA (ab46154), Anti‐Ki67 (ab16667), Anti‐CD31 (ab9498), Anti‐GAPDH antibody (ab8245), and Rabbit Anti‐Human IgG H&L (HRP) (ab6759).
5
Storax Oil Attenuates Inflammatory Response
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Refined storax oil was purchased from Tianjin ZHONGXIN Pharmaceutical Group Limited by Share Ltd Darentang Pharmaceutical Factory and conformed to the standard of China Pharmacopeia (2010 version). Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), glucose-free DMEM, fetal bovine serum (FBS), HBSS, and 0.25% Trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). CytoTox-ONETM Homogeneous Membrane Integrity Assay and Cell Counting Kit-8 (CCK-8) were purchased from Promega (Madison, WI, USA) and Dojindo (Kumamoto, Japan), respectively. DCFH-DA and Trizol were obtained from Invitrogen (Eugene, USA). Penicillin and streptomycin were purchased from Beyotime (Shanghai, China). Anti-NF-κB p65, anti-p-IκBα antibody, anti-p-IKK antibody, and anti-β-actin antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-LaminB1 antibody, anti-iNOS antibody, anti-IL-1β antibody, anti-ICAM-1 antibody, and anti-VCAM-1 antibody were purchased from Abcam Technology (Cambridge, MA, USA). SYBR® Select Master Mix and High Capacity cDNA Reverse Transcription Kits were obtained from Applied Biosystems (Foster City, USA).
6
Comprehensive Analysis of Aortic Atherosclerosis
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Atherosclerotic lesions was examined as we reported previously.25 In brief, mice were euthanized with an overdose of pentobarbital and perfused with 0.9% sodium chloride solution. The heart and aorta were removed immediately. The thoracic aorta was opened longitudinally, and the abdominal aorta was snap‐frozen in liquid nitrogen for further analyses. The severity of abdominal aortic aneurysm was determined as reported previously.26 Atherosclerotic lesions in the aortic arch were determined by en face Sudan IV staining. The characteristics of atherosclerotic plaques were examined in frozen sections of lesions in the aortic root (at 5‐μm intervals). Lipid deposition in plaques was determined by oil red O staining. The expression of vascular cell adhesion molecule‐1 (VCAM‐1) and intercellular cell adhesion molecule‐1 (ICAM‐1), and accumulation of macrophages in plaques were examined by immunohistochemical staining. Sections were incubated with anti‐VCAM‐1 antibody (Abcam, Cambridge, MA), anti‐ICAM‐1 antibody (Abcam), or anti‐Mac3 antibody (BD Biosciences, Bedford, MA), followed by the avidin‐biotin complex technique and stained using a Vector Red substrate kit (Vector Laboratories, Burlingame, CA). Each section was counterstained with hematoxylin.
7
Evaluating Leukocyte Transmigration in DSS-Induced Colitis
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To test whether an increased hemovascular leukocyte transmigration, rather than a reduced lymphovascular leukocyte clearance, could be responsible for the aggravated inflammatory phenotype and amplified intestinal immune cell burden seen in DSS-treated FoxC2(+/−) mice, the colonic expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) was evaluated in DSS-treated WT and FoxC2(+/−) mice. Colon samples were prepared as described above, immunoblotted to either nitrocellulose (ICAM-1) or polyvinyl difluoride membranes (VCAM-1) and incubated overnight with anti-ICAM-1 (1 µg/mL, Abcam) or anti-VCAM-1 antibodies (1:2000, Abcam). Band intensity (densitometry) relative to actin was quantified using NIH Image-J analysis program (imagej.net ).
8
Quantifying ICAM-1 and VCAM-1 Expression in HUVECs
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We added rWRS to the wells of an eight-part slide culture of HUVECs at 5 µg/mL in serum-free HuMedia-EG2 medium. After 12 h of incubation at 37 °C, the cells were fixed with paraformaldehyde. After washing the cells with PBS and then blocking them with 1% fetal calf serum, they were treated with monoclonal anti-ICAM-1 antibodies (1:200; Abcam, Tokyo, Japan) and anti-VCAM-1 antibodies (1:200; Abcam) overnight at 4 °C. The cells were washed with PBS and then treated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000; Thermo Fisher Scientific), followed by the staining of the nuclei with DAPI (1:1000; Thermo Fisher Scientific). We analyzed the treated cells using fluorescence microscopy (KEYENCE, Osaka, Japan) [61 (link)]. The fluorescence intensity was measured using the image processing software ImageJ.
9
Evaluating Leukocyte Transmigration in DSS-Treated Mice
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