Vt 1000 s microsystem

Rats were decapitated and the brains were quickly removed, fixed with 4% neutral buffered formalin for 24 h, and cut into 50-µm thick slices on a vibratome (Leica VT 1000 S Microsystem, Germany). The slices were incubated for one night with the goat anti-mouse NG2 antibody (1:500; ab 50009, Abcam, Cambridge, United Kingdom) and goat anti-rabbit GFAP antibody (1:500; ab 207165, Abcam, Cambridge, United Kingdom). In the next day, after several rinses in phosphate-buffered saline, the slices were incubated for 1 h with 130 µl fluorescent-labeled secondary antibodies (goat anti-mouse IgG (H+L) Alexa Four 647; goat anti-rabbit IgG (H+L) Alexa Four 488; Invitrogen, Molecular Probes, Eugene, Oregon, USA). The slices were analyzed using a confocal microscope (Leica SP5, Germany). Approximately 8–12 slices per animal from cortical and subcortical (excepting hypothalamus and choroid plexus where BBB is leaky) regions were imaged.

Confocal microscopy was performed to confirm an increased BBB permeability to Evans Blue dye. Rats were decapitated and the brains were quickly removed, fixed with 4% neutral buffered formalin for 24 h, and cut into 50-µm thick slices on a vibratome (Leica VT 1000S Microsystem, Germany). The slices were incubated for one night with the goat anti-mouse NG2 antibody (1:500; ab 50009, Abcam, Cambridge, United Kingdom) and goat anti-rabbit GFAP antibody (1:500; ab 207165, Abcam, Cambridge, United Kingdom). In the next day, after several rinses in phosphate-buffered saline, the slices were incubated for 1h with 130 µl fluorescent-labeled secondary antibodies (goat anti-mouse IgG (H+L) Alexa Four 647; goat anti-rabbit IgG (H+L) Alexa Four 488; Invitrogen, Molecular Probes, Eugene, Oregon, USA). The slices were analyzed using a confocal microscope (Leica SP5, Germany). Approximately 8–12 slices per animal from cortical and subcortical (excepting hypothalamus and choroid plexus where the BBB is leaky) regions were imaged.