F3777

Brain sections were immunostained with anti-laminin γ1 (Abcam, AB3297, 1:200), anti-laminin-111 (Sigma, L9393, 1:1000), anti-laminin α2 (Sigma, L0663, 1:200), anti-laminin α4 (R&D system, AF3837, 1:200), anti-AQP4 (Millipore, AB3594, 1:500), anti-occludin (Invitrogen, 71-1500, 1:500), anti-ZO-1 (Invitrogen, 40-2200, 1:500), anti-Claudin-5 (Invitrogen, 34-1600, 1:500), anti-CD31 (BD Pharmingen, 550274, 1:200), anti-PDGFRβ (eBioscience, 14-1402-82, 1:200 and Cell Signaling, 3169S, 1:200), anti-CD13 (BD Pharmingen, 558744, 1:200), anti-Cre recombinase (Novagen, 69050, 1:2000), or anti-Ki67 (Millipore, AB9260, 1:1000) antibodies overnight at 4°C, followed by fluorescent secondary antibodies (Invitrogen) for 1 hour at room temperature. FITC-conjugated SMA (Sigma, F3777, 1:1000) was used to study pericyte differentiation and distinguish capillaries and large vessels, given that SMA is expressed in arterioles and arteries but not capillaries under normal conditions. After mounting, the brain sections were examined and photographed with an Axiovert 200 (Zeiss) microscopy or Leica confocal microscopy. For quantification, at least 9 random images (from at least 3 animals) with GFP or Cre signal at 200X magnification were taken and the mean fluorescence intensity was analyzed.

Thin frozen sections (5 μm) were immunolabeled and quantitation of immunolabeling was performed as previously described(34 (link)). Antibodies related information for stainings on mouse tissues and cells: PGC1-a (1:200; clone 4C1.3, Calbiochem™ ST1202), Twist (1:200; Santa Cruz Biotechnology, inc. H-81), CK8 (1:200; DSHB TROMA-I) and αSMA (1:200; clone 1A4, Sigma-Aldrich® F3777).

HLMFs were grown on 8-well chamber slides and serum-starved for 24 h prior to the experiment. The cells were left unstimulated and incubated in the presence of 0.1% ethanol vehicle control or LXA₄ at 10⁻¹⁰ M and 10⁻⁸ M for 48 hours. Cells were then immunostained as described previously(27 (link)) using FITC-conjugated mouse monoclonal anti-αSMA (F3777, 10 μg/ml, Sigma) and isotype control FITC-conjugated mouse IgG_2a (X0933, 10 μg/ml, Dako, Ely, UK). Cells were mounted with fluorescent mounting medium and cover-slipped. Original images were captured on an epifluorescent microscope (Olympus BX50, Olympus UK Ltd, Southend-on-Sea); grey scale intensity was examined using Cell F imaging software (Olympus UK Ltd). Matched exposures were used for isotype controls.
Actin stress fibres were calculated using a specialised macro on image J designed by Dr Kees Straatman, University of Leicester(15 ). The macro is capable of providing a quantitative, unbiased score of the number of stress fibres per individual cell by determining the fluctuations of grey scale intensity created by the αSMA staining within the stress fibres.

Mouse aortic tissues were harvested and embedded in optimal cutting temperature (OCT) compound, and aorta segments at 6 μm were prepared as described previously.^{16 (link),17 (link)} The serial sections were stained with antibodies for macrophages (Mac-3, 1:900, 553322, BD Biosciences, San Jose, CA), T cells (CD4, 1:90; 553043, and CD8, 1:100, 01041D, BD Biosciences), major histocompatibility complex class-II MHC-II) (1:250, 556999, BD Biosciences), smooth muscle cell (SMC) (α-actin, 1:750, F3777, Sigma-Aldrich), CD31 (1:1500, 553370, BD Biosciences) and neutrophils (Ly6G, 1:200, BE0075, BioXcell, West Lebanon, NH). CD4⁺ and CD8⁺ T cells, and CD31⁺ microvessels were counted blindly and presented as numbers of total cells per mm² of lesion area. Ly6G⁺ neutrophils were counted and presented as number of cells per section. MHC-II-positive and Mac-3-positive areas were measured using computer-assisted image analysis software (Image-Pro Plus; Media Cybernetics, Bethesda, MD) and presented as positive area per mm² of lesion area. Media SMC accumulation was graded according to the grading keys described previously.^{32 (link)}