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.