Srp8033

Samples of liver tissues were excised, and fixed in 10% formalin for 24 h at room temperature. Liver tissues were embedded in paraffin, cut into 5 μm thin sections, and subsequently deparaffinization, rehydration, antigen retrieval and 5% normal goat serum blocking for 4 h at room temperature. The slides were incubated in specific primary antibodies for 4 h, and followed by PBS washing for three times. Then, slides were incubated with horseradish peroxidase‐conjugated secondary antibodies for 2 h, and visualized using substrate diaminobenzidine. The following primary antibodies were used: CD68 (ab125212, Abcam, 1:800 dilution), F4/80 (LS‐C96373‐100, Lifespan, 1:800 dilution), IL‐1β (SRP8033, Sigma, 1:800 dilution), TNF‐α (ab6671, Abcam, 1:1000 dilution). One sample from each mouse was detected, and three high‐power fields at 200× were analyzed for each slide using a microscope (Leica). The number of positive cells were counted.

For DREADDs and TeNT mice, EE, locomotor activity, food intake, and body temperature were monitored by Comprehensive Lab Animal Monitoring System with Temperature Telemetry Transmitter (CLAMS; Columbus Instruments, with G2 E-Mitter transponders). The data were acquired at a 10-min interval per data point as shown in the figures. Temperature transponders were implanted into the peritoneal cavity 3 to 5 days before testing. Stimuli (drugs or temperature) were delivered between 10 a.m. and 3 p.m. (for long-time stimuli, it may occur between 2 p.m. to 8 p.m.) in the dark phase. Mice were adapted in the metabolic chambers for 2 days before giving saline [volume (μl) = 10 × body weight (grams)], CNO (0.1 to 2.5 mg/kg body weight, i.p.; ENZO, no. BML-NS105-0025, as indicated in each figure), CL316243 (1 mg/kg body weight, i.p.; Merck, no. 5.04761.0001), IL-1β (3 μg/kg body weight, i.p.; Sigma-Aldrich, no. SRP8033).