Oct compound

Heart and the ascending aorta were dissected. The detailed method was described previously [51 (link)]. Briefly, the heart and ascending aorta were embedded in OCT compound (CellPath, Oxford, UK), frozen and cut in 10 μm-thick serial cryosections. Eight sections were collected at 100 μm intervals starting at a 100 μm distance from the appearance of the aortic valves. Sections were fixed and stained with oil-red-O (Sigma-Aldrich, St. Louis, MO, USA) and examined under an Olympus BX50 (Olympus, Tokyo, Japan) microscope. Images of the aorta were recorded using an Olympus Camedia 5050 digital camera and stored as TIFF files of resolution 1024 × 768 pixels. The area of the lesion was measured using LSM Image Browser 3 software (Zeiss, Jena, Germany). For each animal, a mean lesion area was calculated from eight sections, reflecting the cross-section area covered by atherosclerosis.

In vitro and in vivo constructs were fixed with 1% (w/v) PFA for 2 h, followed by 25% (w/v) sucrose incubation for 12 h. Samples were then embedded in optimal cutting temperature (OCT) compound (CellPath, Newton, UK), frozen in freezing isopentane, and cryosectioned (12 μm thickness). Cryosections were incubated for 1 h in 0.3% w/v Triton X-100 and 2% v/v normal goat serum, or 5% v/v donkey serum in PBS (blocking buffer), and then for 1 h in the following primary antibodies: mouse anti-PECAM-1 (CD31, endothelial cells, 1:100, LubioScience Gmbh, Zürich, Switzerland), rabbit anti-Ki67 (proliferating cells, 1:100, Abcam, Cambridge, UK), rabbit anti-phospho-histone H3 (Ser28) (1:400, Cell Signaling Technology, Danvers, MA, USA) and rabbit anti-cleaved caspase3 (cells in apoptosis, 1:100, Cell Signaling, Technology Inc., Danvers, MA, USA), mouse monoclonal anti-Human Nuclei (HuNu) (clone 235-1, human cells, 1:100, Merck Millipore, Burlington, MA, USA), and goat anti-Ve-Cadherin (endothelial cells, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA). Tissue sections were then incubated in the dark for 1 h in fluorescently labeled Alexa488 and Alexa546 secondary antibody (dilution 1:200, Thermo Fisher Scientific Inc., Waltham, MA, USA). Cell nuclei were stained with 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI, 300nM, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Animals were treated in compliance with the Swiss Federal guidelines for animal welfare and all procedures were approved by the Veterinary Office of the Canton Bern (Bern, Switzerland) and conform to the Directive 2010/63/EU of the European Parliament.
Eight-week-old male nude rat (Hsd: RH-rnu/rnu, Envigo, weight range 250 ± 16 g) were anesthetized by inhalation using a mixture of oxygen (0.6 L/min) and isoflurane (1.5–3 vol %). Four grafts were implanted in distinct subcutaneous pockets in the back of each rat (4 samples per experimental groups for three different donors). At the moment of sacrifice after 3, 7 and 28 days post-implantation, the total rat vasculature was perfused with 1% w/v paraformaldeide (PFA) following anesthesia by intraperitoneal injection of a mixture of ketamine (100 mg/Kg) and xylazin (10 mg/Kg). The harvested constructs were further fixed in PFA 1% w/v for 30 min and leaved in sucrose 30% w/v overnight at 4 °C before embedding in OCT compound (CellPath), and frozen in isopentane vapors cooled in liquid nitrogen. In some experiments, fluorescein isothiocyanate (FITC)-labeled Lycopersicon esculentum lectin (250 μg in 250 μl; Vector Laboratories) was injected into the femoral vein and allowed to circulate for 4 min before sacrifice in order to label perfused vessels as previously described^{57 (link)}.

In situ lung neutrophil infection was analyzed in mice on day 1 or 23 after infection with 5x10⁶ CFU of the green fluorescent BCG strain Myc409 [7 (link)]. Freshly collected lungs were incubated overnight at 4°C in 4% paraformaldehyde, 10% sucrose (w/vol in PBS) and then immersed in 30% sucrose (w/vol in PBS) for 4 h. Lung tissue was snap frozen in OCT compound (CellPath, Powys, UK). 8 μm-thick lung sections collected on Superfrost Plus slides (Thermo Fischer Scientific) were air-dried and stored at -80°C. Cryosections were blocked for 30 min with Fc-blocking antibody 2.4 G2 (BD Biosciences), washed in PBS with 2% FCS and incubated overnight at 4°C with anti-Ly-6G (clone1A8), (BD Biosciences) followed by incubation with Alexa 594 conjugated anti-rat (Invitrogen) and anti CD3ε (145-2C11, APC conjugated, Miltenyi Biotec) for the localization of T cells and neutrophils. Slides were washed in PBS with 2% and mounted with Fluoromount-G (eBioscience) including DAPI. Images were captured with a confocal Leica TCS SP8 microscope.

For eAT and liver, fresh tissue were fixed with 4% paraformaldehyde for at least 48 h, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) (G1003; Wuhan servicebio technology Ltd., Wuhan, China). Adipocyte size and liver steatosis score were assessed using Image Pro Plus v6.0 (Media Cybernetics Inc., Silver Springs, MD, USA). For each mouse, mean areas of adipocytes were determined in at least five discontinuous scans under ×100 magnification, and scores of liver steatosis were assessed as described previously [34 (link)].
For analysis of atherosclerotic lesion in aortic sinus, the proximal aorta attached to heart was harvested, fixed in 4% paraformaldehyde for at least 48 h, embedded in optimal cutting temperature (OCT) compound (4583; CellPath Ltd., Newtown, Powys, UK) and frozen at −20 °C. From the beginning of aortic root to the extent for 200 μm, four sections (8-μm thickness) taken at 50 μm intervals were collected from each mouse. Sections were stained with Oil Red O (O0625; Sigma-Aldrich Ltd., St. Louis, MO, USA). The area of atherosclerotic lesion in the aortic sinus was measured using Image Pro Plus v6.0 and expressed as the mean size of the four sections for each mouse.

Two or four hours following intracerebral or intravenous injection of rrIL-1β or saline, mice were anaesthetized with pentobarbital (20% w/v pentobarbital sodium, i.p.; J M Loveridge Ltd, UK) and then transcardially perfused with cold 0.9% saline with heparin (Sigma, UK). Liver and striatum were frozen on dry ice for mRNA extraction. The striatum was dissected into ipsilateral (injected) and contralateral side from saline and IL-1β injected animals, and the contralateral (right) side from naïve animals. Alternately, mice were transcardially perfused fixed with 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) after 0.9% saline with heparin (Sigma, UK). Brain and liver were collected and immersed into 4% paraformaldehyde at 4 °C for 24 h, before cryoprotection in 30% sucrose (4 °C) and subsequently embedded in O.C.T. compound (CellPath, UK). Ten-micrometer-thick sections were cut on a cryostat Leica CM1850 (Leica Microsystems, UK) and mounted on gelatin-coated slides in a serial manner. Brain sections were collected for the full extent of the injection site and lesion, from approximately Bregma + 1.10 to − 0.10, based on The Mouse Brain in Stereotaxic Coordinates (Paxinos and Franklin).