Mouse anti cd147

Cell lysis, protein sample preparation and Western blot were carried out as previously described [27 (link)]. Briefly, primary antibodies mouse anti-MCT1 (1:500, sc-365501, Santa Cruz Biotechnology), rabbit anti-MCT4 (1:500, sc-50329, Santa Cruz Biotechnology), mouse anti-CD147 (1:500, sc-71038, Santa Cruz Biotechnology), mouse anti-HIF-1α (1:500, 610958, BD Biosciences) and goat anti-actin (1:500, sc-1616, Santa Cruz Biotechnology) were used. Membranes were then incubated with the adequate secondary antibodies coupled to horseradish peroxidase (Santa Cruz Biotechnology) and bound antibodies were visualised by chemiluminescence (Supersignal West Femto kit, Pierce, Rockford, IL, USA). Protein quantification was performed using ImageJ Software (version 1.41).

Cultured astrocytes were prepared as described above, Astrocyte cultures. After washing with Dulbecco's PBS, the cells were lysed. The lysates were then resolved on a 12.5% (w/v) sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene fluoride membranes. Next, the membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T) and 4% (w/v) skim milk before being incubated overnight at 4°C with the following primary antibodies: mouse anti-HIF-1α (1:400; Novus Biologicals), rabbit anti-MCT1 (1:400; Novus Biologicals), rabbit anti-MCT4 (1:300; Novus Biologicals), mouse anti-CD147 (1:100; Santa Cruz Biotechnology), mouse anti-Na⁺/K⁺-ATPase α1(1:1,000; Santa Cruz Biotechnology), or mouse anti-β-actin (1:10,000; Sigma-Aldrich). After three washes with TBS-T, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse antibody (1:10,000; GE HealthCare) or anti-rabbit antibody (1:10,000; GE HealthCare). The membranes were then washed three times with TBS-T, and the proteins were visualized using the Chemi-Lumi One Ultra system (Nacalai Tesque). Images were obtained using an LAS-4000 imager (Fujifilm).

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).