Mouse anti ng2

The rats were anesthetized with chloral hydrate (400 mg/kg) and then transcardially perfused with saline followed by 4% paraformaldehyde (PFA) (pH 7.4). The brains were removed, post-fixed in 4% PFA, and immersed in cryoprotectant solution (30% sucrose in 0.1 M phosphate buffer) until the tissue sank. Tissues were frozen in dry ice and stored at −80 °C until they were used. Twenty-micrometer frozen coronal sections were cut using a cryostat. The slides were blocked with 5% goat serum in PBS containing 0.1% Triton X-100 for 1 h at room temperature (RT) and then incubated with primary antibodies for 2 h at RT. After being washed, the slides were then incubated for 2 h at RT with secondary antibodies. The following primary antibodies were used in this study: mouse anti-GFAP (1:500, Millipore), rabbit anti-Iba1 (1:1000, Wako), mouse anti-CD11b/c (1:500, DB Pharmingen), mouse anti-NG2 (1:200, Millipore), rabbit anti-cannabinoid receptor 2 (1:100, Abcam), and rabbit anti-rat TSPO (1:200, Biobyt). The following secondary antibodies were used in this study: Alexa Fluor 488 anti-rabbit-IgG and Alexa Fluor 594 anti-mouse-IgG (1:500, Invitrogen).

Immunofluorescence staining was performed on free-floating sections (25 μm) for tissues or glass coverslips for cell cultures in 24-well plates. Primary microglia, neurons, and oligodendrocytes grown in 24-well plates were fixed with 4% paraformaldehyde. Slides or glass coverslips were washed in PBS and immersed in monkey serum (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) for 30 min. Primary antibodies included the following: rabbit anti-MAP2 (sc-20172, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rat anti-CD16/32 (553142, BD, Franklin Lakes, NJ, USA), goat anti-CD206 (AF2535, R & D Systems, Minneapolis, USA), rabbit anti-inducible nitric oxide synthase (iNOS, ab15323, Abcam, San Francisco, CA, USA), goat anti-Arg1 (sc-18351, Santa Cruz Biotechnology), rabbit anti-Iba1 (019-19741, Wako, Osaka, Japan), mouse anti-NG2 (MAB5384, Millipore, Billerica, MA, USA), and rabbit anti-MBP (ab40390, Abcam). The nuclei of cells were stained with DAPI (4′6-diamidino-2-phenylindole; Invitrogen) before taking images. Sections or cells were observed under a fluorescence microscope (Carl Zeiss, Jena, Germany) or confocal microscopy (Leica, Wetzlar, Germany).