Gelatine from porcine skin

Hearts were perfused with 30 mL of a fluorescent gel, containing FITC fluorophore (Fluorescein Isothiocyanate) conjugated with Bovine Serum Albumine (BSA), recently developed by Tsai et al.^{18 (link)} and previously used to stain brain capillary network^{15 (link)}. The gel contains 2% of Gelatine from Porcine Skin (no. G1890; Sigma-Aldrich) in PBS. Once melted, the solution was filtered, added with 0.05% of BSA-conjugated FITC (Albumin-fluorescein isothiocyanate conjugate, Sigma-Aldrich) and filtered again. The preparation was kept at 37 °C in gentle shaking until use. Gel solidification into the vessels was favoured by keeping the organ in contact with iced water for 30 min.
For vascular network labelling of non-fixed hearts, the organ has been perfused with 50 mg of a dextran (Dextran fluorescein; Molecular Probes), solubilized in 2 mL of Tyrode Solution. Images were immediately acquired on left and right ventricles.

Gelatine from porcine skin (Sigma Aldrich, St. Louis, MO, USA) was added to warm Dulbecco’s phosphate buffer saline (DPBS) (Thermo Fischer, Waltham, MA, USA) at a concentration of 100 mg mL⁻¹ and stirred until fully dissolved at 50 °C. When the gelatine solution was clear, methacrylic anhydride (MAA, Sigma Aldrich) at a concentration of 8% (v/v) was added dropwise. After this, the solution was left stirring for 3 more h at 50 °C. The solution was then diluted 1:5 in DBPS to reach a final gelatine concentration of 20 mg mL⁻¹. Gelatine methacrylate (GelMa) solution was placed in 2000 MWCO dialysis tubes (Sigma Aldrich) and dialysed in ultrapure water at 45–50 °C. Water was changed three times a day for 1 week. Once GelMa solution was dialysed, it was placed at −80 °C for 24 h, lyophilised and stored until further used.

Gelatine from porcine skin (Sigma Life Sciences, St. Louis, MO, USA) was dissolved in warm serum-free media or PBS, magnetically stirred until fully dissolved, and filtered twice (20 $\mathsf{\mu }$ m filter). Hydrogels were cross-linked by UV irradiation (90 min).