Mm af morphometric analysis software

The corneal cauterization assay was performed, as previously described [28 (link)]. Eight-week-old female C57BL/6 mice were anesthetized with a mixture of xylazine (10 mg/kg) and ketamine (100 mg/kg). After application of a local anesthetic (Unicaïne, 0.4%) to the eye, the cornea was thermally cauterized using an ophthalmic cautery. One day after cauterization, drops of 10 µL of CXCL9(74-103) (100 µg/mL) or PBS were applied daily for 4 days. On day 5, the mice were sacrificed, and the corneas were removed. Fixation and blocking of whole-mounted corneas were performed in 70% (v/v) ethanol for 1 h and 3% (w/v) BSA in PBS for 1 h, respectively. To stain for blood vessels, the corneas were incubated with rat anti-mouse CD31 antibody (clone MEC13.3, BD Biosciences) overnight and AlexaFluor568 goat anti-rat secondary antibody (Cat No A-11077, Invitrogen) for 2 h. Corneas were flat mounted on microscope glasses overlaid with Prolong Gold antifade mounting medium and imaged using a Leica DMI6000 microscope (Leica Microsystems, Wetzlar, Germany). The cornea blood vessel area was quantified using Leica MM AF morphometric analysis software (Leica Microsystems) and expressed as the percentage of the total corneal area. Ethical approval for animal experiments was obtained from the Ethical Committee of KU Leuven (number LA1210604).

Choroidal neovascularization (CNV) was induced by laser burns using a Purepoint Laser (Alcon, Fort Worth, USA)^{67 (link)}. Ten or eight impacts (for choroidal endothelial cell isolation or histological analysis, respectively) rupturing the Bruch’s membrane were made around the optical nerve using a laser diameter of 100 μm, power 0.320 W, and exposure time of 0.05 s in both eyes. On day 7, at the height of the angiogenic response^{56 (link)}, mice were euthanized by cervical dislocation (three mice (six eyes) per condition), and the eyes (for histological analysis) were enucleated 10 min after retrobulbar injection with Fluorescein isothiocyanate (FITC)-conjugated dextran (Mr 2,000,000) (Sigma-Aldrich) and fixed in 2% paraformaldehyde. Choroids were dissected, flat-mounted (ProLong Gold antifade reagent, Thermo Fisher Scientific), and imaged using a Leica TCS SPE confocal microscope (Leica Microsystems). Analysis of the neovascular area was performed with the Leica MM AF morphometric analysis software (Leica Microsystems) and expressed as the FITC-dextran positive area in percent of the total CNV lesion area. This procedure was repeated for 4–5 independent replicate experiments, each: three mice (six eyes) per condition.