2.5 Immunofluorescence
Deeply anesthetized mice were perfused with ice-cold saline and 4% paraformaldehyde and the brain was dissected out. After post fixation with 4% paraformaldehyde for 24 h, the brains were immersed in 15% sucrose and 30% sucrose. 6 μm or 20 μm cryosections were obtained by slicing with Cryostat Microtome (Leica CM1800, Leica Microsystems GmbH, Germany). For immunofluorescence, slices were permeabilized with 0.5% Triton X-100 and blocked with 2% bovine serum albumin, followed by incubating overnight at 4 ◦C with primary antibodies involving: anti-neuronal nuclei (NeuN; Proteintech, Chicago, IL), anti-ionized calcium-binding adapter molecule 1 (Iba1; Abcam, Cambridge, UK), anti-glial fibrillary acidic protein (GFAP; Proteintech), anti-microtubule-associated protein 2 (MAP2; Abcam), anti-CD31 (Abcam), anti-ZO-1 (Invitrogen, Carlsbad, CA), anti-occludin (Invitrogen), anti-claudin-5 (Invitrogen), anti-intercellular cell adhesion molecule-1(ICAM-1, Santa Cruz, CA, USA), anti-Arg1 (Santa Cruz), anti-CD16/32 (Invitrogen) and anti-TRPM4 (Sigma-Aldrich). Then, slices were washed and incubated with appropriate Alexa Fluor dye of secondary antibodies for 1 h at 37 ◦C. DAPI was used to stain the nucleus. Fluorescence images were randomly obtained with a fluorescence microscopy (LSM 880, Carl Zeiss, Germany) and the digital images were analyzed using ImageJ software blindly by independent investigators.
2.6 Fluoro-Jade C staining
For evaluation of neuronal degeneration, Fluoro-Jade C (FJC) staining was performed with an FJC ready-to-dilute staining kit (Biosensis, USA). Slides were incubated with 1% sodium hydroxide in 80% ethanol for 5 min. After washing with 70% ethanol for 2 min, then with distilled water for 2 min, the slides were incubated with 0.06% potassium permanganate solution for 10 min. Slides were rinsed in distilled water for 2 min, followed by being transferred into 0.0001% solution of FJC which was dissolved in 0.1% acetic acid. Afterward, slides were rinsed in distilled water for 1min in each of 3 distilled water rinses and dried at 60 ◦C for 10 min. Finally, they were cleared in xylene for 1 min and cover slipped with DPX. Quantified analysis was performed with ImageJ software by independent investigators.