Figure 4

Bovine 2D enteroid form tight junctions and a functional epithelial barrier. A Representative confocal images of the adherens junction protein, E-cadherin (green) and B 3X3 tile image demonstrating uniformed expression of the tight junction protein ZO-1 (red) in 2D enteroids seeded at 2.5 × 10⁴ cells/well on Matrigel coated transwell inserts in BEM/1% FBS media. C Z-stack modeling indicate the expression of E-cadherin (green) between the cells and D strong apical expression of F-actin (red) in the 2D enteroids. E EdU (magenta) uptake by E-cadherin⁺ (green) proliferating cells in 2D enteroids. Nuclei are visualised by Hoechst staining (blue). Cells were imaged on Matrigel coated transwell inserts. F Short-term, high TEER can be achieved by seeding 2.5 × 10⁵ cells/well and G a lower more stable TEER can be reached using 2.5 × 10⁴ cells/well. Data is the median of 5 (F) or 12 (E) independent experiments from 5 to 6 calves. Error bars ± 95% Confidence Interval. H Representative brightfield image of the destablistion of the 2D enteroids leading to a drop in TEER on day 12 of culture on transwell inserts.