E selectin

HUVECs treated with 10 ng/ml TNF-α and ± 2.5 μg/ml CCN4 for 24 h were lysed using 5% SDS lysis buffer and equal amounts of protein were loaded onto a gel. After transfer onto a nitrocellulose membrane, 0.061 mg/ml ICAM-1 (Abcam ab53013), 0.437 mg/ml VCAM-1 (Abcam ab134047), 0.5 mg/ml E-selectin (Abcam ab18981) and 0.0635 mg/ml P-selectin (Abcam ab182135) antibodies were added overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at RT and detection was achieved using ECL and a Bio-Rad densitometer. Conditioned media was collected after 24 h and interleukin-6 (IL-6) protein quantified by ELISA (R&D Systems), using the manufacturer’s instructions.Macrophage migration.The peripheral blood mononuclear cells were collected, washed and seeded in 20 ng/ml M-CSF in 10% FCS/RPMI 1640. The resultant adherent monocyte cells were matured by culture for 7 days before use. Macrophages were seeded in a 24-well plate transwell (Millipore, 141006) in the presence or absence of 0.5 μg/ml recombinant CCN4 and with 20 ng/ml MCP-1 in the bottom of the tissue culture plate well. After culture for 48 h migrated macrophages were stained with haematoxylin and quantified.

Proteins were extracted with a lysis buffer containing 0.1% Triton X-100 and a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich), and their concentration was measured by Bradford assay (Bio-Rad). The proteins were separated on (8%–12%) SDS-PAGE and transferred onto polyvinylidene fluoride membrane [13 (link)]. Membranes were probed with primary antibodies against transforming growth factor-β (TGF-β), TGF-β receptor (TGF-βR), SMAD3, tumor necrosis factor α (TNFα), interleukin 6 (IL-6), Nox2, Nox4, E-selectin (Abcam), NF-κB, interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1) (Santa Cruz Biotechnology), eNOS (Thermo Fisher Scientific), and phospho-SMAD3^Ser423/425 (Cell Signaling Technology, Danvers, MA, USA). Loading conditions were determined with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Sigma-Aldrich). Peroxidase-conjugated secondary antibodies were employed to detect primary antibodies (Santa Cruz Biotechnology). Antibody binding was visualized by chemiluminescence, and images were collected and analyzed using a ChemiDoc-It Imager (Ultra-Violet Products, Cambridge, UK).

Formalin-fixed, paraffin-embedded skin samples were deparaffinized with two 30 min washes in xylene, then a series of two 100% ethanol washes for 1 min each, and 1 min washes in 90%, 80%, 70%, 50% ethanol then water. Antigen retrieval was performed in a solution of 10 mM sodium citrate (pH 6.0) or 1 mM EDTA buffer (pH 8.0) for 40 min at 95 °C. Once cooled to room temperature (RT) samples were blocked for 1 h in 5% BSA, 20% donkey serum, and 0.1% Triton-X100 in PBS. Primary antibodies for CD31 (1:50, DAKO, Glostrup, Denmark), E-selectin (1:25, Abcam), ICAM-1 (1:50, Abcam), and vWF (1:200, DAKO) were applied overnight at 4 °C, followed by the corresponding secondary antibody for 1 h at room temperature. Slides were mounted using DAPI-containing mounting media (Vector Laboratories). Slides were imaged using a Zeiss LSM 510 META confocal microscope.

Western blot analysis was performed as described previously43 . In brief, lung tissues were homogenized in ice cold lysis buffer (PBS, 0.5% Triton X-100, pH 7.4) containing protease and phosphatase inhibitors (Roche Complete mini). Lung homogenate was centrifuged (14,000 × g) at 4 °C for 15 min and supernatant was collected for further analysis. Twenty micrograms of protein were loaded onto each well, separated on 10% SDS-polyacrylamide gel and then transferred onto a nitrocellulose membrane (Bio-Rad) using a Bio-Rad Mini-Blot transfer apparatus. Immunoblotting was performed at 4 °C overnight using primary antibodies directed against ICAM-1 (R&D Systems, MN), VCAM-1 (Abcam, Cambridge, MA.), E-selectin (Abcam, Cambridge, MA), VE-cadherin (Cell Signaling, Danvers, MA), β-Catenin (Cell Signaling, Danvers, MA), p-Src (Tyr416) (Cell Signaling, Danvers, MA) and GAPDH (Cell Signaling, Danvers, MA). Membranes were then incubated with a 1:5000 dilution of a secondary antibody (Li-Cor Biosciences, Lincoln, NE) at room temperature for 1 hr. Protein bands were visualized using the Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE).

After deparaffinisation and rehydration, the paraffin embedded heart sections were placed in a 10 mM citrate buffer solution and treated with 3% H₂O₂ in PBS for 5 min. Then, the sections were blocked with 10% normal goat serum for 10 min, and incubated overnight at 4°C with primary antibody [VEGFR2, VCAM-1, ICAM-1, E-selectin (Abcam, Britain), LC3 (CST, USA)] or an equivalent amount of normal goat IgG (CST, USA) as a negative control. After treated with avidin-biotin affinity system for 30 min at room temperature, and stained with 3-3’ diaminobenzidine substrate, the sections were examined under a optical microscope (Olympus X71-F22PH, Japan).. All positive cells were carefully evaluated under double-blind conditions. Image-pro Plus software (Media Cybernetics, United States) was used to calculate the average integrated optical density (IOD) per stained area (μm²) (IOD/area) for positive staining.

2x10⁴ EA.hy926 cells/slide were seeded in Lab-Tek II Chamber Slide^TM (ThermoScientific, USA) and allowed to adhere overnight. Then, cells were incubated with 5 μM simvastatin or 20 μM benznidazole for 24 hours prior to T. cruzi- infection (Dm28c clone) at a MOI of 10. After 16 hours of infection, cells were fixed in 4% formaldehyde–0.1 M phosphate buffer (pH 7.3) for 10 min. Cells were blocked with 3% bovine serum albumin for 1 hour. Then, cells were incubated with monoclonal antibodies against E-selectin (1:100), VCAM-1 (1:250) and ICAM-1 (1:100) (from Abcam, UK) overnight at 4°C. The samples were washed with PBS and incubated with anti-rabbit IgG that had been conjugated with fluorescein (1:100) from Sigma-Aldrich for 1 h. Finally, nuclei were stained with DAPI for 5 minutes and mounted with Dako Fluorescence Mounting (Dako, USA). The cells were photographed using a Nikon Eclipse 400 fluorescence microscope (Nikon, Japan), and images were analyzed by mean intensity using ImageJ software (ImageJ 1.47v).

Cells or tissues were homogenized in ice‐cold suspension buffer supplemented with a proteinase inhibitor cocktail (Sigma‐Aldrich) as described previously.25 Protein concentrations were determined using the BCA Protein assay kit (Thermo Scientific, Waltham, MA). Equal amounts of protein were fractionated by SDS polyacrylamide gels, followed by immunoblotting with the following primary antibodies: MMP1 (AP51345; Abgent); MMP2 (AP51352; Abgent); MMP3 (AP51354; Abgent); MMP7 (ALS11637; Abgent); MMP8 (AP51356; Abgent); MMP9 (AP6214a; Abgent); MMP10 (AP50649; Abgent); MMP11 (AP51347; Abgent); MMP12 (AP6196a; Abgent); MMP13 (AP51348; Abgent); MMP14 (AP6198a; Abgent); TIMP1 (sc‐21734; Santa Cruz Biotechnology); TIMP2 (sc‐365671; Santa Cruz Biotechnology); TIMP3 (sc‐373839; Santa Cruz Biotechnology); TIMP4 (sc‐9375; Santa Cruz Biotechnology); intracellular adhesion molecule‐1 (ICAM1; ab124759; Abcam); VCAM1 (ab134047; Abcam), E‐selectin (ab18981; Abcam), P‐selectin (ab178424; Abcam); nuclear factor kappa B (NF‐κB) Pathway Sampler Kit (9936; Cell Signaling Technology [CST], Danvers, MA); and phospho‐MAPK (mitogen‐activated protein kinase) Family Antibody Sampler Kit (9910; CST). Membranes were then incubated with peroxidase‐conjugated secondary antibody, and specific bands were detected with a Bio‐Rad (Hercules, CA) imaging system.