Aqua poly mount media

Hydrogels were fixed in 2% neutral buffered formalin for 2 hours. Subsequently, for wholemount analyses, fixed hydrogel samples were permeabilized and blocked in 0.25 % Triton X-100 with 1% BSA in 1X PBS. Next, the hydrogel samples were incubated with primary antibodies in 0.5X blocking buffer, rabbit anti-EZRIN (1:100 Cell Signaling, MA), sheep antiCD31 (1:50, R&D Minneapolis, MN), rabbit anti-COL4 (1:100, Abcam, UK), goat anti-MITF (1:100 Santa Cruz, TX), rabbit anti-PLVAP (1:100, Sigma-Aldrich, St. Louis, MO), mouse anti-VE-CADHERIN (1:100, Santa Cruz, TX), mouse anti-ZO1 (1:20, Thermo Fisher Scientific, MA),overnight at 4 °C. The next day, following 2 hours of washes in 1X PBS containing 0.25 % Triton X-100 and 1% BSA, hydrogel samples were incubated in host-specific Alexa-Fluor conjugated antibody (1:500, Thermo Fisher Scientific, MA) in 0.5X blocking buffer and Hoechst (1:2000, Thermo Fisher, MA) at 4°C overnight. Subsequently, the hydrogel samples were subjected to overnight washes and mounted with AQUA-Polymount media (Polysciences Warrington, PA) on depression slides (fisher scientific, NH). All images were captured on a confocal microscope (LSM 510 META, Zeiss, Thornwood, NY).