G6642

Mice were deeply anesthetized and transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in PBS. For fixation, the brains were kept overnight in 4% paraformaldehyde. Each brain sample was placed in 30% sucrose in PBS for 48 h. After embedding and freezing, each brain was cut into 30 µm coronal slices using a cryostat (CM1950, Leica, Heidelberger, Germany).
For immunostaining, the brain slices were washed three times with PBS and incubated with primary antibodies (Foxp1: rabbit anti-Foxp1, 1:20,000, ab16645, Abcam, USA; GABA: rabbit anti-GABA, 1:1000; PA5-32241, Invitrogen, USA; Orexin: mouse anti-orexin-A,1:600, sc-80263, Santa Cruz Biotechnology, USA; MCH: rabbit anti-melanin-concentrating-hormone, 1:1000, M8440, Sigma-Aldrich, USA; Glutamate: rabbit anti-glutamate, 1:1000, G6642, Sigma, USA; and Biocytin: 1:1000, S21374, Invitrogen, USA) dissolved in PBST (0.3% Triton X-100 in PBS) overnight at 4 °C. The next day, slices were washed with PBS and incubated with secondary antibodies (Donkey anti-rabbit/goat, 1:1000; Jackson ImmunoResearch, USA) for 2 h. Fluorescence images were captured using a confocal microscope (Nikon AIR-MP).

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).

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).