Alexa fluor 555 647

Four percent paraformaldehyde (Sigma–Aldrich) was used to fix hNTOs for 2 h followed by washing with PBS. Permeabilization and blocking solutions were made with 0.3% Triton X (PanREAC AppliChem) and 0.5% BSA solution (Sigma–Aldrich) for 30 min. Primary antibodies (Supplementary Data 3) were suspended in permeabilization and blocking solution and applied to the fixed and permeabilized hNTOs for 24 h. Three PBS washes were performed over an additional 24 h period. Subsequently, immunolabeling was performed over a duration of 24 h using donkey-anti mouse Alexa Fluor 555/647 (Invitrogen, dilution 1:500), donkey-anti rabbit Alexa Fluor 555/647 (Invitrogen, dilution 1:500), donkey-anti goat Alexa Fluor 555/647 (Invitrogen, dilution 1:500) and donkey-anti rat Alexa Fluor 647 (Jackson ImmunoResearch, dilution 1:500) secondary antibodies. To visualize filamentous actin we employed Alexa Fluor 647 conjugated phalloidin (Abcam, dilution 1:500). Hoechst was used to visualize nuclei. Click-it EDU Alexa Fluor 647 kit (Invitrogen) was used to label cells with active DNA synthesis. To visualize ZO1, the ZO1 reporter cell line was used.

Mouse embryos were fixed at 4°C with Stefanini solution; 20 g/L Paraformaldehyde, 6.667% saturated picric acid and equal amounts of Sörensen Buffer 0.2M, pH7.2 and distilled water, followed by equilibration in 20% sucrose prior to 10 μm cryo-sectioning. Sections were permeabilized in PBS/0.1% Triton X-100, blocked with 10% goat or donkey serum (Sigma-Aldrich) and stained with primary antibodies: chicken anti-mouse eGFP (Millipore), rabbit anti-mouse MYL2 (Synaptic Systems), rat anti-mouse ITGA6 Allophycocyanin (APC) (eBioscience), rat anti-mouse ITGA5 Biotin (LifeSpan Biosciences) and rabbit anti-mouse cTropT PE (BD Biosciences). Primary antibodies were visualized with secondary antibodies conjugated to FITC/Cy3/Cy5 (Jackson ImmunoResearch) or Alexa Fluor 555/647 (Invitrogen). Hoechst 33342 (Invitrogen) was used to localize nuclei. Imaging was performed using a Zeiss LSM 780 (Germany) laser scanning confocal microscope or a Leica DM500 B (Switzerland).