Ma5 13188

To observe migration, morphology, and angiogenesis of the embedded HUVEC, F-actin, CD31, and nuclei were stained using rhodamine-phalloidin, anti-CD31, and DAPI, respectively. First, the formulated scaffold was fixed with 4% paraformaldehyde (P6148, Sigma-Aldrich, U.S.A.) for 40 min at room temperature (RT). The fixed scaffold was immersed in alginate lyase (Sigma-Aldrich, U.S.A.) solution to remove alginate at 37°C. The alginate removed HUVEC core was immersed in a collagen matrix and incubated at 37°C for gelation. Subsequently, the HUVEC core in collagen was permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, U.S.A.) for 5 min at RT. Primary antibody of anti-CD31 (MA5-13188, Invitrogen, U.S.A.) was incubated at 4°C overnight. Then, secondary antibodies (Alexa Fluor 488, Invitrogen, U.S.A.) and Phalloidin (Alexa Fluor 488, Invitrogen, U.S.A.) were applied for 2 h at RT. Besides, nuclei of the HUVEC core were stained with DAPI (D1396, Invitrogen, U.S.A.) for 5 min. After every chemical treating step, the treated sample was washed 3 times with PBS for 5 min. The stained samples were observed using an IX53 inverted fluorescent microscope (Olympus, Japan) and a FV1000 laser scanning confocal microscope (Olympus, Japan).

After 28 days of culture, Unpass cells were fixed with 1% PFA for 1 hour and washed twice with PBS. For human native adipose tissue, samples were fixed overnight with 1% PFA and washed twice with PBS, embedded in paraffin wax, and sectioned (7 μm). Antibodies against T-cad at 1:100 (#ABT121, Sigma), vWF at 1:500 (#M0616, Dako), CD31 at 1:250 (#MA5-13188, Invitrogen), and Laminin at 1:250 (#33-530, Invitrogen) were used as primary antibodies. Fluorescent-conjugated secondary antibodies chicken anti-rabbit Alexa 488 (#A-21441, Thermo Fischer Scientific) and goat anti-mouse Alexa 546 (#A-11030, Thermo Fischer Scientific) were both used at 1:500. Images were acquired with a Nikon A1R confocal microscope.