Mouse anti mct4

Cultured astrocytes were prepared as described above, Astrocyte cultures. The cells were plated in a 35-mm µ-Dish (ibidi) and treated with 10 µM BzATP for 24 h. The cultured astrocytes were then fixed with 4% (w/v) paraformaldehyde for 30 min, permeabilized with 0.3% (v/v) Triton X-100 for 10 min, and treated with 3% (w/v) bovine serum albumin in PBS for 30 min to block nonspecific binding. Next, the cells were incubated for 2 d at 4°C with the following primary antibodies: rabbit anti-MCT1 (1:300; Novus Biologicals) or mouse anti-MCT4 (1:50; Santa Cruz Biotechnology). The cells were washed before being incubated for 1 h at room temperature with the following secondary antibodies: Alexa 546-conjugated anti-mouse or anti-rabbit IgG (Invitrogen). The nuclei were then counterstained with 4′,6-diamidino-2-phenylindole solution (Nacalai Tesque). Fluorescence images were obtained using a confocal laser scanning microscope.

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).