Ab119774

Immunohistochemical staining was performed on 4-μm serial cryosections of frozen cardiac tissue from the right atrium. Immunocytochemistry was performed on formalin-fixed human cardiomyocytes and cardiac endothelial cells cultured under normoxic or hypoxic conditions for 6 h. Mouse monoclonal antibodies against ALOX15 (ab 119774, 1:100, Abcam) in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA), with isotype control, mouse IgG 2b (GR128531-1, Abcam). Goat anti-mouse IgG (H+L) Alexa Fluor 594 (A11032, 1:200, Molecular Probes, USA) in PBS with 1% BSA was used as secondary antibody. Antibody staining was examined with a Zeiss Axioplan 2 Imaging microscope.

IL-1β–stimulated AT and AR tendon stromal cells were grown in chamber slides and treated as previously described. After incubation in 15-epi-LXA₄ or MaR1, cells were fixed in ice-cold methanol for 5 min and washed with PBS. Immunofluorescence staining protocols and image acquisition are adapted from a previously published protocol (23 (link)). Tendon stromal cells isolated from AT and AR donors (n = 3 each) were incubated with the following primary antibodies: anti-ALX (ab26316; Abcam, Cambridge, MA, USA), anti–arachidonate lipoxygenase (ALOX) 15 (ab119774; Abcam), anti-ALOX12 (ab211506; Abcam), anti–Phospho-STAT-1 (ab29045; Abcam), anti-PDPN (ab10288; Abcam), and anti–IL-6 (ab9324; Abcam) in PBS containing 5% goat serum in saponin for 3 h at room temperature. For negative controls, the primary antibody was substituted for universal isotype control antibodies: cocktail of mouse IgG₁, IgG_2a, IgG₂b, IgG₃, and IgM (Agilent Technologies, Santa Clara, CA, USA) and rabbit Ig fraction of serum from nonimmunized rabbits, solid phase absorbed. Isotype control staining is shown in Supplemental Fig. S1. Immunofluorescence images were acquired on a Zeiss Laser Scanning Microscope (LSM; Carl Zeiss, Oberkochen, Germany) 710 confocal microscope using a previously described protocol (23 (link)).

Cell lysates of macrophages, corresponding to 3 × 10⁶ cells, were separated on 10% (5-LOX, 15-LOX-1) and 16% (FLAP) polyacrylamide gels, and blotted onto nitrocellulose membranes (Hybond ECL, GE Healthcare, Freiburg, Germany). The membranes were incubated with the following primary antibodies: mouse monoclonal anti-15-LOX-1, 1:500 (ab119774, Abcam, Cambridge, UK); rabbit polyclonal anti-5-LOX, 1:500 (1550 AK6, provided by Dr. Olof Radmark, Karolinska Institutet, Stockholm, Sweden); rabbit polyclonal anti-FLAP, 0.1 μg/ml (ab85227, Abcam, Cambridge, UK); rabbit anti-β-actin, 1:1000 (4967S, Cell Signaling, Danvers, MA). Immunoreactive bands were stained with IRDye 800CW Goat anti-Mouse IgG (H + L), 1:10,000 (926-3221, LI-COR Biotechnology, Cambridge, UK) and/or IRDye 680LT Goat anti-Rabbit IgG (H + L), 1:40,000 (926-68023, LI-COR Biotechnology, Cambridge, UK), and visualized by an Odyssey infrared imager (LI-COR Biosciences). Data from densitometric analysis were background corrected.

Biopsies were attached onto a cork disc, embedded in the tissue-mounting reagent Tragacant (Histolab, Gothenburg, Sweden), snap frozen in liquid nitrogen, and preserved at -80°C until cryosectioning. Frozen tissue was sectioned into 7-μm serial sections using a cryotome. Immunohistochemistry (IHC) was conducted using 7-μm cryosections of left ventricular tissue. Frozen tissue sections were fixed in -20°C acetone for 10 min, washed with phosphate-buffered saline (PBS) solution, and blocked in PBS containing 2% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO), 0.3% Triton–X100 (Sigma-Aldrich), and 5% goat serum (Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes at room temperature. Samples were stained using ALOX15 mouse IgG2b monoclonal (ab119774, Abcam, Cambridge, UK) or ALOX15B rabbit IgG polyclonal (ab23691, Abcam) antibodies diluted in Tris-buffered saline with 1% BSA. Corresponding isotype controls for the primary antibodies were used for determining background staining. Results were visualized by staining with secondary antibodies: goat anti-mouse IgG Alexa Fluor 647 (A11032, Thermo Fisher Scientific) and donkey anti-rabbit IgG Alexa Fluor 546 (A10040, Thermo Fisher Scientific) for ALOX15B, diluted in PBS with 1% BSA. Samples were mounted with prolong gold antifade with DAPI (Thermo Fisher Scientific).

Human cardiomyocytes and cardiac endothelial cells were incubated under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions for 6 h. Total cellular lysates were prepared using RIPA buffer supplemented with a mammalian protease inhibitor cocktail (Sigma-Aldrich). Immunoblots were prepared as described [3 (link)]. The membranes were probed with anti-ALOX15 antibody (ab 119774, Abcam) or anti-β-actin (A5441, Sigma-Aldrich). The relative intensity of the protein bands was quantified using the ImageLab software (BioRad).