Prolonged gold anti fade mounting media

The iPSC-derived BMECs were seeded in 8-well chamber slides and treated as mentioned earlier. Cells were quickly washed with ice-cold PBS and fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA, USA) for 10 min and blocked for 30 min at room temperature (RT) in the presence of PBS supplemented with 10% goat serum (ThermoFisher) and 0.2% Triton-X100 (Sigma). Cells were incubated overnight at 4 °C in primary antibodies targeting claudin-5 (1:100), occludin (1:100), and ZO-1 (1:100) diluted in 10% goat serum (PBSG). After washing three times with PBS, cells were incubated for 1 h at room temperature with Alexa Fluor conjugated secondary antibody in the dark. Finally, cells were washed thrice with PBS and mounted in prolonged gold anti-fade mounting media (Invitrogen, OR, USA). Mounted slides were examined using multi-photon confocal microscopy (Ti-E, Nikon, NY, USA).

HCMEC/D3 cells were seeded in two-well chamber slides and grown as mentioned earlier. Cells were fixed with 16%, methanol free formaldehyde (diluted 1 in 4 in 1X PBS; from Polysciences Inc. # 18814) after specified experimental exposure duration. This was followed by three PBS washes and cell permeabilization using 0.02% Triton 100X. After another three PBS washes, fixed cells were blocked with 5% goat serum in PBS (blocking buffer) at room temperature (RT) for 45 min and incubated overnight at 4°C with primary antibodies prepared in blocking buffer. The following day, cells were incubated for 1 h at RT with Alexa Fluor^® 488 or 555 conjugated goat anti-rabbit or anti-mouse antibodies or vice versa, respectively (1:1,000) after three washes with PBS. Thereafter, cells were rinsed, dried and mounted with DAPI in prolonged gold anti-fade mounting media (invitrogen). Mounted slides were examined with EVOS digital inverted fluorescence microscope after overnight drying. Cell slides stained with only secondary antibodies served as negative controls.