Immunohistochemical analyses were carried out as previously described [31 (link)33 (link)]. Briefly, mice were anesthetized with a mixture (3.5:1) of ketamine hydrochloride (50 mg/ml) and xylazine hydrochloride (23.3 mg/ml) at a dose of 2.5 µl/g body weight. They were then perfused with 4% paraformaldehyde by a trans-cardiac method, and brains were isolated. Brains were post-fixed further in the same solution overnight at 4℃. Brains were coronally cut into 40-µm-thick sections with a vibratome (Leica VT 1000S; Leica Instruments, Nussloch, Germany). These sections in a floating state were incubated with 4% bovine serum albumin in PBS with Tween-20 (PBST) for 1 h, then reacted with the primary antibody at 4℃ overnight. The sections were washed with PBST, reacted with the secondary antibody, and visualized with an ABC Elite kit (PK-6200, Vector Laboratories, Burlingame, CA, USA) for DAB staining.
Anti-MCH was purchased from Phoenix Pharmaceuticals (#H-070-47; 1:500; Germany), while the secondary antibody, biotinylated anti-rabbit IgG, was purchased from Vector Laboratories (#BA-1000; 1:200, Burlingame, CA, USA). DAB-stained images were analyzed with an Olympus BX 51 microscope equipped with a DP71 camera and MetaMorph Microscopy Automation & Image Analysis software (Molecular Devices, Sunnyvale, CA, USA).
Anti-MCH was purchased from Phoenix Pharmaceuticals (#H-070-47; 1:500; Germany), while the secondary antibody, biotinylated anti-rabbit IgG, was purchased from Vector Laboratories (#BA-1000; 1:200, Burlingame, CA, USA). DAB-stained images were analyzed with an Olympus BX 51 microscope equipped with a DP71 camera and MetaMorph Microscopy Automation & Image Analysis software (Molecular Devices, Sunnyvale, CA, USA).