Alexa fluor 488 goat anti rabbit immunoglobulin

Brains of each group (n = 3) at day 3 and day 7 were fixed with fresh 4% paraformaldehyde solution, and cut into coronal sections of 10 μm thickness for immunofluorescence staining of claudin‐5 (1:200, 34‐1600, Invitrogen) at 4°C for overnight. Subsequently, brain samples were washed with PBS and incubated with the Alexa Fluor^® 488 goat anti‐rabbit immunoglobulin (Life Technologies) secondary antibodies for 1 hour at 37°C. Every group was digitally photographed with a confocal laser scanning microscope (Leica Microsystems Inc., Buffalo Grove, United States).

Every fifth slice of a total section was used to detect activations of microglia in ipsilateral hemisphere by incubation with primary antibody against Iba1 (1 : 200, Abcam, UK) and anti-CD206 (1 : 500, Abcam, UK) at 4°C for overnight. BV2 cells in six groups seeded on slides were fixed with 4% paraformaldehyde for 30 min at 4°C, permeabilized with 0.3% Triton X-100 for 15 min, and incubated with anti-P65 (1 : 500, CST) overnight. Both brain tissue and cell slides were washed with PBS and, respectively, incubated with the bs-0294D-FITC (1 : 200, Beijing Biosynthesis Biotechnology Co., Ltd., CHA) and Alexa Fluor® 488 goat anti-rabbit immunoglobulin (1 : 200, Life Technologies) secondary antibodies for 1 h at room temperature. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI; Sigma). The number of BV2 with positive signal of nuclear P65 was calculated in five randomly selected microscopic fields at 400× magnification. Fluorescent labeling of Iba1, CD206, and TUNEL were observed with the Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems Inc., Buffalo Grove, USA), and five nonoverlapping fields of one slice in the penumbral cortex were measured. Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) was used for quantitative analysis.