Bz x800 system

Isolated epididymal white adipose tissue (eWAT) and livers were fixed in 4% formaldehyde for 24 h and embedded in paraffin. Six-micrometer-thick sections were stained with hematoxylin & eosin (H&E) and then used in subsequent analyses. Regarding CD11c immunohistochemistry, paraffin-embedded sections were incubated with a hamster anti-mouse CD11c antibody (dilution 1:100, 10 μg/mL) for 3 h followed by a goat anti-hamster IgG antibody (dilution 1:100, 8 μg/mL) for 1 h. Photomicrographs were captured using the microscope BX61 (Olympus, Tokyo, Japan). Isolated gastrocnemius muscles were quenched with hexane and frozen. Tissue was embedded in OCT compound (Sakura Finetek, Osaka, Japan), and 20-µm-thick cross-sections were obtained from the center of muscle tissue using a cryostat and stained with H&E. Photomicrographs were captured using BZX800 (Keyence, Osaka, Japan). The cross-sections of each adipocyte and myofiber were traced one by one, and the area of each traced region was analyzed, using ImageJ 1.45 s software (NIH) or the BZX800 system (Keyence), respectively [29 (link), 31 (link)]. The average size of adipocytes and myofiber was calculated by analyzing ~ 300 adipocytes or myofibers per mouse from the H&E-stained sections of eWAT or gastrocnemius muscle.

Mice were treated with isoflurane and transcardially perfused with phosphate buffered saline (PBS) and 4% paraformaldehyde (PFA). Brains were collected and immersed in 4% PFA for 2–4 h at room temperature (RT). Fixed brains were transferred to 20% and 30% sequentially sucrose for dehydration. Dehydrated brains were imbedded in Cryo-Embedding Compound (Cat# 16362, Ted Pella Inc., Redding, California, USA) and sectioned into 20~40-µm-thick slices with cryostat (HM550, Thermo Scientific). Slices were washed with PBS (30 min), permeabilized with PBST (0.5% Triton X-100 in PBS, 30 min) and blocked with 10% donkey serum (in PBST, 1 h, RT). Incubation of primary antibody (in 10% donkey serum) was performed overnight at 4 °C. After washing with PBS (10 min × 3 times), slices were incubated in secondary antibody (2 h, RT). After wash with PBS, slices were mounted onto slides and sealed with SlowFade® Diamond Antifade Mountant (cat. 36972, ThermoFisher). Antibodies used included: chicken anti-GFP (1:2000, Cat# GFP-1020, AVES, RRID:AB_10000240); rabbit anti-TH (1:1000, Cat# MAB318, Chemicon, RRID:AB_2201528). Images were captured with Zeiss LSM 800 confocal microscope. AAV expression at LDTg and dorsal raphe nucleus (DRN) was analyzed by the Keyence BZ-X800 system.