Ab26316

fMLP-R expression was evaluated in each layer (NDNs and LDNs). Prior to staining, 10⁶ cells were harvested and washed twice in PBS 1X.
Granulocytes were resuspended in blocking buffer (PBS 1X containing 2% FBS) and incubated on ice for 20 min. The cell suspension was then permeabilized with 0.3% Triton X-100 and incubated with anti-FPRL1 antibody [GM1D6] (2 mg/mL; ab26316, Abcam, Germany) for 45 min. Cells were washed three times in washing buffer and incubated in dark for 45 min with secondary goat Alexa594-coupled anti-mouse IgG antibody (1:500 in washing buffer, Invitrogen) and 50 μg/ml of 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories). Cells were then washed twice in washing buffer and suspended in 500 μL PBS 1X, for finally being mounted in a drop of ProLong Antifade reagent (P36930; Thermo Fisher Scientific).
Images were taken using an Axio Imager M.1 microscope (Zeiss) and analyzed using Zen software (Fig. 5A). A library of images was randomly established in order to have at least 200 granulocytes for each slide. Cells were identified as neutrophils based on nuclear morphology (segmentation of the nucleus) and because of the paucity in eosinophils and basophils (data not shown) by an operator blinded to horses and to the layer.

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)).