Sp8 inverse confocal laser scanning microscope

Widefieled image acquisition was done on a Zeiss AxioObserver Z1 widefield microscope, equipped with a Lumencor SpectraX illumination system and a Hamamatsu Orca Flash 4.0 V2, sCMOS, cooled fluorescence camera (16 bit, 2048 × 2048 pixel (4 MP), pixel size 6.5 µm). A 63×, 1.4-NA, i-plan apochromat oil-immersion objective was used. For optimal representation in figures, images were adjusted for brightness and exported as RGB TIF files using Fiji^{53 (link)}.
Confocal images were acquired with a Leica SP8 inverse confocal laser scanning microscope with a 63×, 1.4-NA Plan-Apochromat oil-immersion objective. The sequential scanning mode was used and the number of overexposed pixels was kept at a minimum. Fields were recorded at a resolution of 512 × 512 pixels, 8 bit depth or 1024 × 1024 pixels, 8 bit. For optimal representation in figures, maximum intensity projections were calculated and images were adjusted for brightness and exported as RGB TIF files using Fiji^{53 (link)}.

At the terminal time point, cells cultured either on glass, Collagen I, or hTEMs were washed with PBS and fixed in 4% formalin for 15–20 min. Afterwards, cells were permeabilized 10 min with 0.1% Triton-X-100 in PBS, again washed in PBS, and blocked with a blocking buffer composed of 2% BSA in PBS for 1 h. Primary antibody for Vascular Endothelial Cadherin (Ve-Cad), Collagen III, Von Willebrand Factor (vWF), Collagen IV, Phalloidin, and Vinculin (Supplementary Table S2) were solubilized in a 1% BSA/PBS solution and incubated for 1 h at room temperature. After washing with 0.1% Tween-20 in PBS and PBS, samples were incubated with the corresponding secondary antibodies (Supplementary Table S2) for 1 h at room temperature. Finally, cells were again washed with 0.1% Tween-20/PBS and PBS and embedded with the Fluoroshield Mounting Medium with DAPI (Abcam). The stained cells were visualized using a Leica SP8 inverse confocal laser scanning microscope (DMI6000 AFC). Image processing was performed using the LAS AF Lite Microscope software (Leica, version 4.0.11706).