Immunofluorescence Staining of Tendon Cells

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)).