Rat tail collagen 1 gel

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).

Aortic ring assay was performed as described 91 , 92 (link). Aorta from the postnatal day 3 (P3) C57BL/6J mice was cut into 1 mm rings before being embedded in a 96-well plate coated with rat tail collagen I 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 µL of OptiMEM supplemented with 2% FBS and 1× 1× penicillin and streptomycin overnight. The following day, media was replaced with OptiMEM supplemented with 2% FBS and 1× penicillin and streptomycin containing 50 ng/mL VEGF with different concentrations of EGCG, accordingly. Treatment media were changed every other day. At day 10 of culture, the explants were fixed in 4% PFA and stained with Griffonia Simplicifolia Lectin (GSL) isolectin IB4 (Vector Lab, ZB0406) and AN2-PE (Miltenyi Biotec, 130-100-468). Vessel outgrowth was visualized under the Eclipse Ti-E Inverted Research Microscope. The number of sprouts was counted manually.