Ab31630

The cryosections of the aortic root were fixed in 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). The sections were blocked using 5% normal goat or donkey serum (Abcam) at room temperature for 2 h and then incubated with primary antibodies, including anti-CD68 (1:100, Abcam, ab31630), anti-VCAM-1 (1:100, Abcam, ab134047), anti-E-Selectin (1:100, Santa Cruz, sc-137054), overnight at 4 °C followed by appropriate fluorescence-conjugated secondary antibodies (ThermoFisher, Waltham, MA, USA) for 2 h at room temperature in the dark. Nuclei were stained with Hoechst 33342 (ThermoFisher, Waltham, MA, USA) and mounted with a fluorescence mounting medium (Electron Microscopy Sciences, Cat#17985-10, Hatfield, PA, USA). Images were acquired using an Olympus FV1200 confocal microscope (Olympus Corporation, Tokyo, Japan). Quantification of immunofluorescence staining was performed with the Image J software.

After SW imaging, heart weight was measured to calculate relative heart weight against body weight. The heart was sectioned transversely at the mid-papillary level, and then fixed with 10% formalin, embedded in paraffin, and cut into 5-μm sections. Sections were stained with hematoxylin–eosin for evaluating infiltrating inflammatory cells, and with picrosirius red for evaluating fibrosis. Macrophages, which accounted for the majority of infiltrating inflammatory cells in EAM model, were identified by mouse anti-rat CD68 monoclonal antibody (ab31630, Abcam) staining. The CD68-positive area was quantitatively calculated using ImageJ software (version 1.52v, National Institutes of Health, Bethesda, MD, USA) by setting an intensity threshold that matched the visually identified staining areas as previously reported^{5 (link)}. Similar to SW imaging, the percentage of CD68-positive area in a circular ROI of 1-mm in diameter was measured at five locations, and the average value was calculated. The percentage of CD68-positive area was compared between each group. The relationship between SWDS and the percentage of CD68-positive area was analyzed to examine whether SWDS reflects pathologically evaluated myocardial inflammation.

Implant neutrophil and macrophage numbers were determined by staining for Myeloperoxidase (MPO; ab90810, Abcam) and CD68 (ab31630, Abcam) respectively. Cellular localisation of NGAL and MMP-9 in implant inflammatory cells and isolated peripheral blood neutrophils was determined using antiNGAL (M145, SantaCruz) and antiMMP-9 (sc-6840, SantaCruz) antibodies according to standard procedures [19 (link)]. The secondary antibodies (Alexa Fluor 488 or 594, Invitrogen) were used for visualisation and the nuclei were stained with DAPI. Images were captured using an Olympus AX-70-Fluorescence microscope.
The cellular localisation of NGAL (ab41105; Abcam) and MMP-9 (ab76003; Abcam) was examined in skin wound tissue by immunohistochemistry. For quantitation of NGAL, the staining intensity was examined in the epithelial (top), granulation tissue (middle) and inflammatory (basal) zones and scored by two independent observers based on a scale of 0 = no staining above isotype control to 3 = intense staining. The score for each zone was summed and the data was analysed using Chi squared analysis at an intensity cut off at ≥ 6. MMP-9 staining was localised mainly to cells in the granulation tissue layer and the staining intensity in this area was quantified using Image J software.

The liver and lung were dissected free and fixed in 10% formalin solution. The sections were stained with Hematoxylin and Eosin (H&E) and examined by light microscopy. Liver sections were stained with Sirius Red (Polysciences Inc., Warrington, PA, U.S.A.) to determine the extent of collagen deposition. The immunohistochemical staining was performed with anti-CD68 antibody (diluted 1:200, ab31630, Abcam Cambridge, UK) to detect pulmonary CD68-positive macrophages, with anti-CD31 antibody (1:200; Serotec and Pharmingen, San Diego, CA) to determine hepatic angiogenesis, and with anti-von Willebrand factor (vWF) antibody (1:100, MCA127T, AbD Serotec, UK) to determine pulmonary angiogenesis [4 (link),17 (link)]. The numbers of CD68-, CD31-, and vWF-positive cells per high-power field (magnification 200x) were counted by a semi-quantification method [18 (link)].

Four-micron-thick brain coronal sections were prepared as described earlier, deparaffinized in xylene, rehydrated via alcohol gradients, and washed with PBS (0.01 M, pH 7.4). Following antigen retrieval, the sections were blocked in 5% bovine serum albumin for 30 min at room temperature (RT) and incubated with primary antibodies as follows: rabbit anti-activated Notch1 (1:200; Ab52301, Abcam, Cambridge, UK); goat anti-Iba1 (1:300; Ab5076, Abcam); mouse anti-CD68 (1:200; Ab31630, Abcam); rabbit anti-NeuN (1:200, 24307T, Cell Signaling Technology, Danvers MA, USA) overnight at 4 °C. The next day, the slices were washed with PBS and incubated with secondary antibodies: donkey anti-goat Alexa 488 (1:500; A11055, Invitrogen), donkey anti-rabbit Alexa 555 (1:500; A31572, Invitrogen), donkey anti-mouse Alexa 488 (1:500; A21202, Invitrogen), donkey anti-goat Alexa 555 (1:500; A21432, Invitrogen), goat anti-mouse Alexa 555 (1:500; A21422, Invitrogen), and donkey anti-rabbit Alexa 488 (1:500; A21206, Invitrogen) for 1 h at RT. After washing with PBS three times, the sections were stained by 4′6-diamidino-2-phenylindole for 10 min at room temperature and observed under a fluorescence microscope (Leica-DMI8, Leica Microsystems, Wetzlar, Germany).