Anti digoxigenin rhodamine fab fragments

To perform 3D-FISH, hPSCs and their differentiated counterparts were cultured on coverslips, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature (RT), washed in PBS (3× for 5 min), permeabilized with 0.5% Triton X-100 in PBS for 30 min, equilibrated in 2× SSC, and treated with RNase A (10 μg/ml, Qiagen) at 37°C for 1 hour. After washing the cells again in 2× SSC and PBS, they were incubated in 0.1 N HCl, permeabilized with 0.2% saponin/PBS and 100 mM Tris-HCl, and briefly rinsed in 2× SSC. Cellular DNA was treated with a hybridization mixture (70% formamide/2× SSC) at 75°C for 5-6 min and dried in an ethanol series. After DNA probe denaturation (75°C for 7 min) and reannealing (37°C for 30 min), the probes were mixed with Hybrisol VII (Qbiogene, Carlsbad, California, USA), applied to cells, sealed under coverslips with rubber cement, and allowed to hybridize overnight. After hybridization, the slides were washed in 50% formamide and 2× SSC at 45°C and then incubated with anti-digoxigenin-rhodamine Fab fragments and avidin fluorescein conjugate (both purchased from Sigma-Aldrich) for 1 h at 37°C. After rinsing the cells with 2× SSC/0.1% Tween, the coverslips were mounted in DAPI-containing Mowiol (Sigma-Aldrich).

Dihydroethidium (DHE), Terminal Transferase from Calf Thymus (TdT), Digoxigenin-11-UTP, Anti-Digoxigenin-Rhodamine Fab fragments, phenylephrine, Acetylcholine, Haematoxylin, Eosin, Oil Red O, and other fine chemicals were purchased from Sigma (Buchs, Switzerland). Primary antibodies including F4/80, α-smooth muscle actin, Von Willebrand factor, appropriate secondary antibodies conjugated to Alexa Fluor 488/546 and Hoechst were obtained from Abcam (Cambridge, UK).