Author response image 1. Confluent human brain endothelial cells form an impermeable barrier.
The human brain endothelial cells (cell line hCMEC/D3) were cultured on Ibidi-slides. After reaching confluence, cells were washed, fixated with 4% formaldehyde and stained with FITC-labeled antibodies specific for A. the tight junction protein Claudin-5 (green). Nuclei were visualized using Hoechst (blue) or B. the adherens junction protein VE-Cadherin (green) and subsequently cells were analyzed by confocal laser scanning microscopy. From the images it is clear that both Claudin-5 and VE-Cadherin are highly present at cell-cell contacts, indicative of tight barrier formation by the brain endothelial cells.