Gtx125988

The mice were intraperitoneally anesthetized using 0.3% sodium pentobarbital solution. Next, they were perfused with 0.9% saline and 4% paraformaldehyde at a fixed rate. The brains were extracted and stored in 4% paraformaldehyde at 4°C overnight and then dehydrated using a 15 and 30% sucrose solution until they sank. Coronal sections, 20 μm in thickness, were cut by a cryostat frozen microtome (Thermo Fisher Scientific, NX50, United States). For immunofluorescence (IF), the sections were warmed to 37°C for 1 h and then washed with PBST 5 times at 10-min intervals. Subsequently, the brain sections were incubated in 10% donkey blocking buffer at 37°C for 1 h and then with primary antibodies, including anti-c-Fos (1:500, rabbit, ab190289, Abcam) or rabbit anti-GABA (1:500, rabbit, GTX125988, GeneTex), at 4°C for 24 h. The sections were then rewarmed to 37°C for 1 h, washed 5 times with PBST, and finally incubated with corresponding fluorophore-conjugated secondary antibodies (1:800) at 37°C for 1 h. They were later washed an additional 5 times with PSBT and incubated with 4,6-diamidino-2-phenylindole at the final stage. To visualize the fluorescence signals, a digital pathological section scanner was utilized.

Mice were deep anesthetized and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA). Brains were post-fixed for 2 h in 4% PFA at 4°C and then dehydrated by 30% sucrose. Subsequently, brains were coronally sectioned at 40 μm using a cryostat microtome (CM1200, Leica, Germany). The sections were washed in phosphate-buffered saline (PBS, pH = 7.4) and then blocked with 5% normal donkey serum in PBS with 0.3% Triton X-100 (PBST) for 2 h at room temperature. Then, the sections were incubated in 2.5% donkey serum with PBST for 24 h at 4°C with guinea pig anti-c-Fos antibody (1:1,000, 226308, Synaptic Systems, Germany), rabbit anti-glutamate antibody (1:500, G6642, Sigma-Aldrich, USA), and rabbit anti-GABA antibody (1:200, GTX125988, GeneTex, USA), respectively. Afterwards, secondary antibodies, including donkey anti-guinea pig Alexa Fluor 488 (1:500, 706-545-148, Jackson ImmunoResearch, USA), donkey anti-guinea pig Alexa Fluor 594 (1:500, 706-585-148, Jackson ImmunoResearch, USA), donkey anti-guinea pig Alexa Fluor 647 (1:500, 706-605-148, Jackson ImmunoResearch, USA), and donkey anti-rabbit Alexa Fluor 488 (1:500, 711-545-152, Jackson ImmunoResearch, USA), were applied for 2 h at room temperature. At last, the slices were washed and mounted in Fluoromount-G (Millipore, USA) and imaged by laser confocal microscopes (FV1200, Olympus, Japan).