Griffonia simplicifolia isolectin b4 ib4

The aortic ring assay was performed as described [60 (link)]. Aorta from P10 wild type and Pparβ/δ^+/− mice were cut into 1mm rings before being embedded in a 96-well plate coated with rat tail collagen I gel (BD Biosciences, USA). Explants were treated with 50ng/mL VEGF and the media changed every other day. Explants from P3 wild type aorta were treated with 100nM of 10h or DMSO vehicle control. At day 10 of culture, the explants were fixed in 4% PFA and stained with Griffonia Simplicifolia isolectin B4 (IB4) (Vector Lab, Burlingame, CA, USA). Vessel outgrowth was imaged using Eclipse Ti-E Inverted Research Microscope (Nikon, Tokyo, Japan). The IB4⁺ areas were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The aortic ring assay was carried out using a modified method as described previously [47 (link)]. Briefly, thoracic aortas were dissected from P3 mice and cut into rings of approximately 1 mm in diameter. A single aortic ring was embedded per 96-well coated with rat tail collagen gel (BD Biosciences, San Jose, CA, USA). Explants were incubated for 30 min at 37 °C to allow for complete polymerization of the collagen gel. After incubation, aortic ring explants were cultured in 100 uL of OptiMEM supplemented with 2% FBS and 1× P/S for 24 h. The following day, media was replaced with OptiMEM supplemented with 2% FBS and 1× penicillin and streptomycin containing 100 μg/mL rhCFB or vehicle control. Media was replaced every 2 days. On day 10 of culture, the explants were fixed and stained with Griffonia Simplicifolia isolectin B4 (IB4) (Vector Lab, Burlingame, CA, USA). Vessel outgrowth was visualized using Eclipse Ti-E Inverted Research Microscope (Nikon, Tokyo, Japan). The IB4⁺ sprouts were counted manually in real-time. The focus was adjusted manually during counting, to ensure vessels sprouting in different planes were counted.