Histology and Immunofluorescence

Aortic root tissue and ARdH 3D constructs (n = 3) were washed with PBS and fixed in 4% PFA for 1 h at RT. Cryoprotection was performed by successive incubation steps in PBS and 5, 10, 20, and 50% glycerol solutions. After 6 washes in 3% sucrose in 0.1 M phosphate buffer, samples were immersed in OCT compound for 1 h (Neg-50, Thermo Scientific) and cryosections of 7 μm were cut on a Leica Cryotome. Sections were stained with Hematoxylin and Eosin (Vector, H-3502), Picro Sirius Red (Abcam, ab150681) and a modified Russell-Movat pentachrome stain (Nordic BioSite, KSC-L53PIA-1) according to the manufacturer's protocol.

For immunofluorescence, cryosections were brought to room temperature for 10 min and then washed with PBS and blocked in TBS Blotto A (SC-2333, Santa Cruz) with 3% BSA, 0.3% Triton X-100 and 1% cold fish gelatin (Sigma, G7041) for 1 h at room temperature, followed by incubation overnight at 4°C with primary anti-human antibodies: CD31 at 1:100 dilution (SC-376764, Santa Cruz), α-SMA at 1:100 dilution (SC-32251, Santa Cruz), VE-Cadherin at 1:100 (14-1449-82, ThermoFisher), vimentin at 1:500 (SC-6260, Santa Cruz) and endothelin-1 at 1:250 dilution (PA3-067, ThermoFisher). The next day, samples were washed and incubated with Alexa Fluor 594 or FITC conjugated secondary antibodies at a dilution of 1:1,000 for 30′ at RT in the dark. Nuclei were stained with DAPI 10 μg/mL (Sigma, D9542) and samples were mounted in Fluoromount-G (Thermo Scientific, Waltham, MA, USA). Images were acquired using a confocal microscope LSM Airyscan (Carl Zeiss AG, Oberkochen, Germany) and a fluorescence microscope (Olympus IX81, Shinjuku City, Tokyo, Japan) equipped with an XM10 camera. Images were processed using ImageJ software. Mean fluorescence intensity (MFI) of images was calculated for each fluorophore and normalized to the number of cell nuclei stained with DAPI (9 images per sample).