Eaat2

For immunohistochemical analysis, cells were fixed with 4% paraformaldehyde (PFA) in 120mM phosphate buffer (PBS), pH 7.4, permeabilized with 0.05% Triton X-100 in PBS, blocked with 10% goat serum in PBS and subjected to immunohistochemistry staining with primary and secondary antibodies diluted in the blocking solution.
For immunolabelling the following antibodies at indicated dilutions were used: anti-Nestin (1:400; BD Bioscience), anti-Sox1 (1:100; R&D Systems), anti-Sox2 (1:200; Abcam), anti-Pax6 (1:200; DSHB), anti-Ki67 (1:200; Vector Labs), anti-TuJ1 (1:400; Covance), anti-Map2 (1:200; Millipore), anti-DCX (1:200; Millipore), anti-GFAP (1:200; Millipore), anti-O4 (1:50, Sigma-Aldrich), anti-GABA (1:200, Abcam), anti-vGlut1 (1:200; Millipore), anti-TH (1:100, Millipore), anti-Synaptophysin (1:100; Millipore), anti-PSD95 (1:200; Invitrogen), anti-vimentin (1:5000; Abcam), anti-S100β (1:1000; Sigma-Aldrich), anti-aquaporin 4 (AQP4, 1:100; Santa Cruz Biotechnology) and anti-excitatory amino acid transporter 2 (EAAT2, 1:100; Santa Cruz Biotechnology), secondary Alexafluorophore-conjugated antibodies (1:1000, Invitrogen). DNA was stained using Hoechst 33258 (1:10000, Invitrogen). All cells expressing a particular marker were counted manually and normalized to the total number of cells.

Brain tissues were lysed with RIPA buffer (150 mmol/L NaCl, 1% Nonidet P‐40, 0.5% deoxycholic acid, 0.1% SDS and 50 mmol/L Tris‐HCl, pH 7.4) containing a protease inhibitor (Roche), and then sonicated for 1 minutes and incubated on ice for 20 minutes. The supernatant was collected and centrifugated at 13,200 g for 15 minutes at 4°C, and protein concentrations were determined using a BCA assay (Thermo Fisher Scientific). Equal amounts of proteins were then loaded on 8%‐12% SDS polyacrylamide gels. The separated samples were transferred to a PVDF membrane and incubated with the primary antibodies against the target proteins and then with the HRP‐conjugated secondary antibodies. The protein bands were visualized by enhanced chemiluminescence (ECL; Millipore) using a bioimaging analyser (Bio‐Rad). The relative intensity of each band was measured using Image J software (rsb.info.nih.gov, by W. Rasband). Primary antibodies used: human GFAP (R&D Systems, 1:1000), EAAT2 (Santa Cruz, 1:1000), Kir4.1 (Millipore, 1:1000), Arginase‐1 (Santa Cruz, 1:1000) and IL‐1β (Santa Cruz, 1:1000).