Anti glutamate antibody

Deeply anesthetized with isoflurane, mice were transcardially perfused with saline followed by ice-cold 4% paraformaldehyde. Brains were post-fixed in paraformaldehyde for 2 h at room temperature and then immersed in 30% sucrose overnight for dehydration. The brains were coronally cut into 40-μm sections on a freezing microtome (Leica CM1900, Germany). Sections containing the LHA were rinsed in phosphate-buffered saline (PBS, pH 7.4) three times for 10 min each and blocked by 5.0% normal donkey serum (NDS) with 0.3% triton-X100 in PBS (PBST) for 2 h at room temperature. Then the sections were incubated with anti-glutamate antibody (1:500, G6642, Sigma-Aldrich, USA) in 2.5% NDS with PBST for 24 h at 4°C. Afterwards, the sections were washed three times with PBS, and incubated with Alexa Fluor 488 (1:500 diluted in 2.5% NDS with PBST; 715–545–150, Jackson ImmunoResearch, USA) for 2 h at room temperature. Finally, the sections were washed, mounted, cover-slipped, and imaged using a laser confocal fluorescence microscope (VS120, Olympus, Japan).

DMEM/F-12, neurobasal medium, and B27 supplement were obtained from Gibco Invitrogen Corporation (Carlsbad, CA, USA). Fura-2 AM and BAPTA-AM were obtained from Dojindo Molecular Technologies, Inc. Sodium hydrosulfide hydrate (NaHS), EGTA, thapsigargin (TG), nifedipine, nimodipine, and mibefradil were obtained from Sigma-Aldrich. Fura-2 AM and TG were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO did not exceed 0.1%. Anti-MAP-2 antibody produced in mouse, Anti-MAP-2 antibody produced in rabbit, anti-CBS antibody produced in mouse, and anti-MST antibody produced in rabbit were obtained from Abcam. Anti-glutamate antibody produced from rabbit was obtained from Sigma-Aldrich. FITC goat anti-rabbit IgG (H+L) and Cy3 goat anti-mouse IgG (H+L) were obtained from Beyotime Biotechnology. Alexa Fluor® 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 594 goat anti-mouse IgG (H+L) were obtained from Invitrogen.

Animals were anesthetized deeply with a mixture of chloralose (35 mg/kg) and urethane (700 mg/kg) and perfused transcardially with saline (150 mL) followed by 4% paraformaldehyde (250 mL) in 0.1 M sodium phosphate buffer (0.1 M PBS; pH 7.4). Brains were rapidly removed and post-fixed in the same fixative at 4 °C overnight, followed by transfer into 20% and 30% sucrose sequentially in 0.1 M PB for at least 3 days. According to Paxinos and Watson’s atlas, coronal medullary sections at 30 μm thick were cut with a microtome (Leica, Deer Park, IL, USA) 1.5–1.7 mm rostral to the obex. For cells, 4% paraformaldehyde was performed for 30 min after the cells were treated with UII or NaHS. After washing in 0.01 M phosphate-buffered saline (0.01 M PBS; pH 7.4) and absorption in 2% BSA, and 0.2% Triton X-100 in PBS, the sections or the cells were incubated with primary antibodies at 4 °C overnight. Anti-MAP-2 and anti-CBS antibodies were purchased from Abcam (Cambridge, UK). Anti-glutamate antibody was purchased from Sigma-Aldrich. Sections or the cells were then washed in PBS and incubated with fluorescent secondary antibodies for 2 h. The sections or the cells were observed using confocal microscopy (Zeiss LSM510, Jena, Germany).