Monoclonal mouse anti gfap

The following antibodies were used: polyclonal rabbit anti-CLOCK (ab3517, Abcam, (Cambridge, UK)), polyclonal rabbit anti-BMAL1 antibody (NB100-2288, Novus Biologicals), monoclonal mouse anti-GFAP (#3670, Cell signaling technology (Danvers, MA, USA)), polyclonal rabbit anti-Caspase-3 (#9662, Cell signaling Technology (Danvers, MA, USA)), and monoclonal mouse anti-β-actin (A5316, Sigma-Aldrich (St Louis, MO, USA)). Fluoroshield™ with DAPI (F6057, Sigma-Aldrich (St Louis, MO, USA)) was used for nuclear staining and mounting. Sections were mounted onto gelatin-coated slides with Canada Balsam (Wako, Tokyo, Japan) following dehydration.

Cells were fixed with phosphate-buffered saline (PBS) containing 4% of paraformaldehyde (PFA, w/v) for 15 min and then were washed three times with PBS. Cells were membrane-permeabilized with 0.2% Triton X-100 in PBS for 20 min. After blocked by 5% bovine serum albumin (BSA) in PBS for 1 h at RT, cells were incubated with primary antibodies overnight at 4°C. Primary antibodies were monoclonal mouse anti-GFAP (1 : 400; Cell Signaling Technology, 3670), polyclonal rabbit anti-GLT-1 antibody (1 : 100; ProteinTech Group, 22515-1-AP), and monoclonal mouse anti-NF-κB p65 (1 : 400; Cell Signaling Technology, 6956). For surface GluA1 immunolabeling, cells were incubated with the primary antibody (anti-AMPA receptor antibody, 1 : 200, Abcam, 183797) without permeabilization after fixation. After washing, the cells were incubated with secondary antibodies (Alexa 488/555-conjugated goat anti-mouse/rabbit anti-IgG (1 : 400, Invitrogen)) for 3-4 h at RT. The cultures were washed three times with PBS and then stained with DAPI for 15 min. The cells were sealed with antifluorescence quenching slides after three washes with PBS. The fluorescent images were acquired by LSM 880 confocal microscopy (Carl Zeiss Corp., Oberkochen, Germany).

The following antibodies were used: polyclonal rabbit anti-NOX2 (ab129068, Abcam, Cambridge, UK), monoclonal mouse anti-NOX2 (NBP1-41012, Novus), monoclonal rabbit anti-HK2 antibody (#2024), monoclonal rabbit anti-LDH-A antibody (#3582, Cell Signaling Technology), monoclonal rabbit anti-LDH-A antibody (#3582, Cell Signaling Technology), polyclonal rabbit anti-COL5A1 antibody (#37304, Cell Signaling Technology), monoclonal mouse anti-GFAP (#3670, Cell Signaling Technology), monoclonal rabbit anti-GFAP (#12389, Cell Signaling Technology), and monoclonal mouse anti-β-actin (A5316, Sigma-Aldrich). Fluoroshield™ with DAPI (F6057, Sigma-Aldrich) was used for nuclear staining and mounting. Sections were mounted onto gelatin-coated slides with Canada Balsam (Wako, Tokyo, Japan) following dehydration.