Embedding compound

After perfusion, brain segments were postfixed in 4% paraformaldehyde for 24 h, and then permeated with 20% sucrose in 0.1 M phosphate-buffered saline (PBS) (pH 7.4) for 24 h and 30% sucrose in 0.1 M PBS for 48 h at 4 °C. Brain segments were frozen in an embedding compound (Sakura Finetek, Tokyo, Japan) on dry ice. They were cut with a cryostat (Leica CM 1100; Leica, Wetzlar, Germany) at a thickness of 30 μm and collected in PBS at 4 °C to be processed immunohistochemically as free-floating sections. The sections were incubated for 48 h at 4 °C with primary antibodies: mouse anti-GFAP (1:2000; Cat. # AB5804, RRID: AB_10062746), rabbit anti-BDNF (1:2000; Cat. # sc-546, RRID:AB_630940). The sections were washed six times with 0.1 M PBS (10 min each) and then incubated for 3 h at room temperature with the secondary antibody: Alexa488- and Alexa546-conjugated mouse- and rabbit-IgGs.
(Cat. # A-11034, RRID:AB_2576217 and Cat. # A11030, RRID:AB_144695). Immunohistochemical images were obtained using a confocal laser microscope (Fluoview1000; Olympus, Tokyo, Japan) and digital images were captured with Fluoview1000 (Olympus).

Mice were anesthetized with 4% (v/v) isoflurane and perfused transcardially with saline, followed by 4% (w/v) paraformaldehyde in PBS. The brains were removed, postfixed overnight in a solution containing 4% (w/v) paraformaldehyde and 4% (w/v) sucrose in PBS, and cryoprotected in solutions containing 10% (w/v) and 20% (w/v) sucrose in PBS for 1 d each. The brains were then frozen in an embedding compound (Sakura Finetek) on dry ice before coronal sections (20 µm) were cut on a cryostat (CM 1100, Leica). The sections were fixed with 4% (w/v) paraformaldehyde for 30 min, permeabilized with 0.3% (v/v) Triton X-100 for 20 min, and treated with 3% (w/v) bovine serum albumin in PBS for 30 min to block nonspecific binding. Next, the sections were incubated for 3 d at 4°C with the following primary antibodies: rabbit anti-glial fibrillary acidic protein (GFAP; 1:1,000; Millipore), mouse anti-MCT1 (1:250; Abcam), mouse anti-MCT4 (1:50; Santa Cruz Biotechnology), or mouse anti-CD147 (1:50; Santa Cruz Biotechnology). After being washed, the sections were incubated for 1 h at room temperature with the following secondary antibodies: Alexa 488- or Alexa 546-conjugated anti-mouse or -rabbit IgG (Invitrogen). Fluorescence images were obtained using a confocal laser scanning microscope (LSM 780, Carl Zeiss, Jena, Germany).