Rabbit anti glutamate

Mice were deeply anesthetized with an 3% isoflurane and perfused with 20 ml ice-cold phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA). Brains were carefully removed and postfixed in 4% PFA for 6–8 h, and cryoprotected with 30% sucrose for 48 h. The brain was 30 μm thick sections were coronally prepared using a freezing microtome (Leica CM1950). For immunofluorescent staining, the sections were incubated with PBS containing 0.3% Triton X-100 for 1 h at room temperature and subsequently allowed to react with primary antibodies (rabbit anti-glutamate, 1:500, Sigma; rabbit anti-c-Fos, 1:1000, Synaptic Systems) at 4˚C overnight. After washing with PBS, the sections were subsequently coupled with the corresponding fluorophore-conjugated secondary antibodies for 1.5 h at room temperature. Finally, after rinsing 3 times in PBS, sections were mounted with DAPI staining. Confocal images were acquired under a 10x or 20x objective with a 1024 × 1024 resolution using the Olympus confocal microscopes (FV3000, Olympus). Double fluorescence labelled cells were manually counted using ImageJ software. The Paxinos and Franklin atlas (2013) was employed to define the regions of interest.

Mice were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.), the chest was opened, and saline and 4% paraformaldehyde were perfused into the systemic circulation through the ascending aorta. Mouse brains were dissected intact, post-fixed with 4% paraformaldehyde overnight at 4°C, and then transferred to a 30% sucrose solution for 1–2 weeks. The brain was cut into 30–40 μm slices using a freezing microtome (Leica 2000, Germany). For spontaneous fluorescence, the slices were washed three times with 0.3% Triton X-100, 15 minutes at a time, and then cover-slipped. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) Fluoromount-G^® (0100–20, Southern Biotech, Birmingham, AL, USA). For co-localized staining, the slices were blocked for 2 h at room temperature and were then incubated for 12 h at 4°C with rabbit anti-glutamate (1:500 dilution, Sigma) and rabbit anti-GABA (1:500 dilution, Sigma). After washing away unbound primary antibodies, the brain slices were incubated with the corresponding Alexa Fluor 594-conjugated secondary antibody for 2 hours (1:1000 dilution; Invitrogen). The brain slices were mounted on slides and then sealed and stained with DAPI. Images were acquired on a multiphoton laser point scanning confocal microscopy system (FV1000, Olympus, Tokyo, Japan).