Rabbit anti mct1

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.

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).