α smooth muscle actin clone 1a4

For histological characterization, engineered vessels were fixed in formalin (Fisher), dehydrated through a graded ethanol series using a Sakura Tissue Processor (Sakura), embedded in paraffin and sectioned at 7 μm thickness. Sections were deparaffinised, rehydrated through a graded ethanol series and stained using haematoxylin & eosin (SigmaAldrich) following the manufacture’s instructions. For immunofluorescence analyses, tissues were cryopreserved in Cryomatrix (ThermoFisher) and sectioned at 20 μm thickness using a cryotome (Leica). Immunostaining was performed as described [17 (link)] using antibodies specific for the EC marker CD31 (clone JC/70A Biolegend, 1:50) and α-smooth muscle actin (clone 1A4 SigmaAldrich, 1:200). Sections were counter-stained with DAPI and imaged with an inverted microscope (Carl Zeiss). Endothelium barrier integrity was analyzed by injecting Evans blue (Sigma Aldrich) at a final concentration of 0.5% in the circulation loop of the bioreactor for 10 min followed by continuous PBS washing for 20 min. Vessels were cut open longitudinally and en face preparations were analysed macroscopically with photo documentation.

The expression of smooth muscle contractile proteins using monoclonal antibodies specific to α‐smooth muscle actin (clone 1A4, 1:1000; Sigma), calponin (clone hCP, 1:1000; Sigma), caldesmon (clone hHCD, 1:500; Sigma), and myosin heavy chain (clone hSM‐V, 1:500; Sigma) was investigated by immunocytochemistry. HRSMC were seeded in 35‐mm Fluorodish^TM cell culture dishes (World Precision Instruments, USA) and incubated as specified for each experiment. The medium was removed and cells were fixed in 4% formaldehyde in PBS for 10 min at room temperature. After washing in PBS, cells were incubated in 3% BSA + 0.1% Triton in PBS for 1 h at room temperature to block non‐specific protein binding and to permeabilize the cells. The primary antibody diluted in PBS was added to each dish and incubated for 1 h at room temperature, followed by washing and incubation for 1 h with anti‐mouse IgG FITC conjugated secondary antibody (F9006, 1:500; Sigma). The dishes were mounted with glass coverslips using mounting medium containing DAPI (Vectashield® Mounting Medium with DAPI, Vector Laboratories Ltd, UK). Images were acquired using a fluorescence microscope (BX51 Olympus, GX Optical, UK).