Mineral oil

In order to synthesize GelMA, dimethyl sulfoxide (Sigma Aldrich, St. Louis, MO, USA) was mixed with 5 g of gelatin (Sigma Aldrich, St. Louis, MO, USA). The mixture was heated up to 50 °C with continuous stirring. After 0.5 g of 4-(dimethlyamino)-pyridine (Sigma Aldrich, St. Louis, MO, USA) was dissolved with the solution, 2 mL of glycidyl methacrylate (Sigma Aldrich, St. Louis, MO, USA) was added to the solution at a constant flow rate of 0.5 mL/min with vigorous stirring. The reaction was kept for two days under a dry N₂ gas environment. And the solution was filtered using a membrane (molecular weight 12,000 to 14,000) with deionized water at 40 °C for 1 week. The deionized water was replaced once a day. A lyophilization-induced aggregated porous solid was obtained and stored at −80 °C. GelMA prepolymer solutions at 3 wt%, 5 wt%, and 8 wt% were prepared. Sodium alginate (Sigma Aldrich, St. Louis, MO, USA) with an average molecular weight between 12,000 and 40,000 was dissolved in deionized water to prepare concentrations of 0.1 wt%, 0.3 wt%, 0.5 wt%, 0.7 wt%, and 1 wt%, respectively. Mineral oil (Sigma Aldrich, St. Louis, MO, USA) and 25 wt% of Span 80 (Sigma Aldrich, St. Louis, MO, USA) were purchased and mixed together to increase the viscosity of the Mineral oil for emulsification.

TIE-1-Igγ1-WT, 2-16A2 or ASP4021 diluted with PBS was subcutaneously administered to SD rats. At 48 h after administration of the antibody, Evans Blue dye dissolved in physiological saline (Sigma) was intravenously administered. Immediately, 20 μl of allyl isothiocyanate (also known as mustard oil; Nacalai Tesque) diluted with 5% mineral oil (Sigma) was applied to one ear, and mineral oil to the contralateral ear. After 30 min, both ears were sampled, weighed, then immersed in 1 mL of formamide, and incubated at 70 °C overnight to extract Evans Blue dye from the ear tissue. The Evans Blue dye concentration was determined from the absorbance (measurement wavelength of 620 nm and control wavelength of 740 nm) of the extract. The amount of leakage per ear weight was calculated by dividing the Evans Blue dye concentration by the weight of the ear. The final amount of leaked Evans Blue dye from each animal was calculated by subtracting the amount of leaked Evans Blue dye from the ear that received mineral oil from the amount from the ear that received mustard oil in the same animal. The amount of leaked Evans Blue dye was used as an index of vascular permeability.

Alizarin red S is a calcium-sensing dye. DPSCs, differentiated towards the osteogenic phenotype, are able to deposit and to induce the mineralization of extracellular matrix rich in calcium phosphate, which can be identified by Alizarin red S. Calcium deposits are detectable for their bright orange-red color.
The DPSCs in each well were rinsed twice with PBS, PBS was discarded and DPSCs were fixed in paraformaldehyde 4% for 15 min at room temperature and then washed with deionized water. Alizarin red S staining solution 40 mM (Merck KGaA, Darmstadt, Germany) was added to each well and probed for 20 min at room temperature (RT) on a shaker. The wells were washed for five times in deionized water. Calcium deposits, stained in orange-red, were dissolved as follows: 10% acetic acid was added under shaking for 30 min. Laminas were scraped, the liquid containing deposits was collected and vortexed in a tube. Previously heated warm mineral oil (Merck KGaA, Darmstadt, Germany) was added, the tube maintained on ice for 5 min and eventually centrifuged at 20,000 g for 15 min. The supernatant was discarded and 10% ammonium hydroxide (Merck KGaA, Darmstadt, Germany) was added. The final solution was analyzed by a spectrophotometric reading performed at 405 nm wavelength.

Housing of mice and in vivo experiments were performed in compliance with the European Communities Council Directive of 24, November 1986 (86/609/EEC) and national and institutional guidelines. Animal care and experimental procedures were approved by the Animal Care Committee of the Institute of Molecular Genetics (ref. 58/2017). Apc^cKO/cKO mice [41 (link)] were obtained from the Mouse Repository (National Cancer Institute, Frederick, MD, USA); Villin-CreERT2 mice [42 (link)] were kindly provided by Sylvie Robine (Institut Curie, Centre de Recherche, Paris, France); Apc^+/Min (C57BL/6J-ApcMin/J) [43 (link)], Ki67-RFP (Mki67tm1.1Cle/J) [44 (link)], Lgr5-EGFP-IRES-CreERT2 (B6.129P2-Lgr5tm1(cre/ERT2)Cle/J) [45 (link)], and Rosa26-tdTomato (B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) [46 (link)] mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The animals were kept under specific pathogen-free conditions. To induce Cre-mediated gene recombination, mice were gavaged with 1 mg tamoxifen (Merck). tamoxifen was dissolved in ethanol (100 mg/mL) and combined (1:9) with mineral oil (Merck) before administration. Mice were sacrificed by cervical dislocation at various time points after administration of a single dose (100 µL) of the tamoxifen/oil solution.

All reagents were of analytical grade. Graphite powder, copper chloride, potassium ferrocyanide and all phenolic standards were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Mineral oil and the electrolyte buffer salts were purchased from Merck (Darmstadt, Germany).
Foodstuffs samples with known antioxidant activity were purchased from local markets and drugstores of the city of Goiânia, Goiás, Brazil.
Assay samples were prepared by sonication in ethanol:buffer (1:1) solution in order to achieve a 10% final concentration. Standards were prepared in suitable solvents in order to get stock solutions of 10 mM final concentration.