Rabbit anti claudin 5

U87 cells were trypsinized, then plated onto 6-well culture plates with suitable culture medium (high-glucose DMEM, 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin) containing 10³ cells. When 80% of the U87 cells were confluent, the complete culture media was aspirated and replaced with fresh EBM-2 medium containing 5% fetal bovine serum. EC monolayers grown on glass coverslips were placed into culture plates and co-cultured for 4 days. The GEC monolayers grown on glass coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After blocking with 2% bovine serum albumin (BSA) in PBS, the cells were incubated with mouse anti-occludin (diluted 1:50; Life Technologies Corporation, Frederick, MD, USA) and rabbit anti-claudin-5 (diluted 1:50; Life Technologies Corporation, Frederick, MD, USA) to visualize the distribution of occludin and claudin-5, respectively. The cells were incubated with rhodamine-labeled phalloidin (diluted 1:250; Sigma-Aldrich) to assess actin filaments. The cells were incubated with mouse anti-RhoA (diluted 1:150; Abcam, Cambridge, UK) and rabbit anti-ROCK II (diluted 1:50; GeneTex, Irvine, California, USA) to analyze their expression. The glass slides were then evaluated using immunofluorescence microscopy (Olympus, Japan).

Cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature and permeabilized with 0.1% saponin 30 min. After several washes in PBS (Phosphate Buffered Saline Calcium) (8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH₂PO₄, 2.86 g/L NaHPO₄ (12 H₂O), pH 7.4), nonspecific sites were blocked for 30 minutes with PBS containing 10% normal goat serum (NGS) (Sigma-Aldrich). Cells are then incubated with primary polyclonal antibody against Claudin-5 (Rabbit anti-Claudin-5, 1/200^e, 34–1600, Life Technology) or against ZO-1 (Rabbit anti -O-1, 1/200^e, 61–7300, Life Technology) diluted in PBS 2% NGS, for 1 hour at room temperature. After several washing steps with PBS, the secondary polyclonal antibody (Goat anti-rabbit Alexa 568, A11036, Molecular probes) diluted at 1/200^e in PBS 2% NGS, was incubated in the dark for 1 hour. Nuclei are stained with Hœchst 33358 and washed with PBS and distilled water.

For immunofluorescence staining, cells or spinal cord sections were fixed in 4% PFA for 15 min and subsequently permeabilized in 0.2% Triton X-100/PBS for 20 min. The samples were treated with 5% bovine serum albumin (BSA) for 1 h at room temperature to block nonspecific binding. The samples were incubated overnight at 4 °C with primary antibody specific for rabbit anti-ZO1 (1:50, Invitrogen), rabbit anti-claudin-5 (1:100; Invitrogen), rabbit anti-occludin (1:50; Abcam), mouse anti-CD31 (1:200, Invitrogen), mouse anti-NeuN (1:500, Abcam), and rabbit anti-GRP78 (1:200, Proteintech). Samples were washed thrice with PBS and incubated with Alexa 594- or Alexa 488-conjugated secondary antibodies (1:200, Jackson ImmunoResearch, USA) for 1 h in the dark. Finally, nuclei were labeled with DAPI and images were obtained under similar exposure time and conditions.