Rabbit anti claudin 5

For NF-κB and Iba1 double immunofluorescence staining, brain sections were fixed with 4% PFA, rinsed with PBS, and treated with Tris-EDTA solution at 100 °C for 30 min for antigen retrieval. For claudin-5 and CD31 double immunofluorescence staining, sections were treated with 1% NaOH in PBS containing 1% H₂O₂ for 20 min for antigen retrieval. Such sections were blocked with 1% FBS containing 0.3% Triton X-100 and incubated with primary antibodies of either mouse anti-NF-κB p65 (1:100, Santa Cruz Biotechnology)/rabbit anti-Iba1(1:500, Wako Pure Chemicals) or rabbit anti-claudin-5 (1:300, Santa Cruz Biotechnology)/rat anti-CD31 (1:300, Dianova, Hamburg, Germany) at 4 °C overnight. Sections were then incubated with AF488- and Cy3-conugated secondary antibodies for 2 h at room temperature, washed with PBS, and mounted with VECTASHIELD^®.

Fullerenol, C60(OH)₂₄ was purchased from BuckyUSA (Houston, TX, USA) and dissolved in the vehicle solution composed of 0.9% NaCl. Fullerenol was diluted with cell culture medium to 1 (0.19 µM), 10 (1.9 µM) and 100 µg/mL (19 µM). IFN-γ was purchased from Miltenyi (Bergisch Gladbach, Germany). TNF-α was obtained from Peprotech (Hamburg, Germany). Mouse anti-alpha smooth muscle actin, rabbit anti-GFAP, rabbit anti-occludin and rabbit anti-VCAM-1 antibodies were acquired from Abcam (Cambridge, UK). Rabbit anti-caspase 3, cleaved caspase 3, erk1/2, phospho erk1/2 and phospho NFκB p65 were obtained from Cell Signaling (Leiden, The Netherlands). Goat anti-NFκB p65 and ICAM-1, and rabbit anti-claudin 5 were from Santa Cruz (Heidelberg, Germany). Rat anti-CD31 was obtained from Serotec (Puchheim, Germany). Rat anti-VE-cadherin was purchased from eBiosciences (Frankfurt, Germany). Rabbit anti-claudin 3 was obtained from Invitrogen (Karlsruhe, Germany). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).