Donkey anti mouse alexa fluor 488

Mice were deeply anesthetized with isoflurane and transcardially perfused with 4% paraformaldehyde followed by 0.9% saline. Brains were postfixed for 2 h in 4% paraformaldehyde at 4 °C and were then serially transferred to 30% sucrose in phosphate-buffered saline (PBS, PH = 7.4). The brains were coronally sectioned every 40 μm using a Leica cryostat. The sectioned brains were washed with PBS for 5 min three times consecutively, and blocked with 5% normal donkey serum in PBS with Triton X-100 for 2 h at 24–26 °C. Primary antibodies, including rabbit anti-glutamate (1:1000, Sigma-Aldrich), mouse anti-orexin A (1:200, Santa Cruz), mouse anti-OxR2 (1:200, GeneTex), rabbit anti-c-Fos (1:1000, Sigma-Aldrich) and rabbit anti-GABA (1:200, GeneTex) were incubated at 4 °C overnight. The slices were washed with PBS. The incubation of secondary antibodies, including donkey anti-rabbit Alexa Fluor 488 (1:400, Jackson ImmunoResearch), donkey anti-mouse Alexa Fluor 488 (1:400, Jackson ImmunoResearch), donkey anti-rabbit Cy3 (1:400, Jackson ImmunoResearch) or donkey anti-mouse Cy3 (1:400, Jackson ImmunoResearch), were then incubated for 2 h at 24–26 °C. The brain slices were washed with PBS again after incubation, and sections were mounted in Fluoromount-G (Millipore) and imaged using a confocal microscope (FV1200, Olympus).