Plasma and Tissue Analysis in Mice

Mice were euthanized by CO₂ following the recommended procedure of the American Veterinary Medical Association (AVMA Euthanasia Panel Report). Plasma was obtained from blood samples collected with Microvette CB 300LH (SARSTEDT, Nümbrecht, Germany). Tissues were resected, snap-frozen, and stored at −80°C until further analysis. Plasma triglyceride, NEFA, ketone bodies, ALT, insulin, FGF21, albumin and haptoglobin concentrations were determined using commercial assay kits (Serum Triglyceride Determination Kit, Sigma; NEFA-HR (2 (link)), Wako Chemicals; Autokit Total Ketone Bodies, Wako Chemicals; Ultra-Sensitive Mouse Insulin ELISA Kit, Crystal Chem; Mouse/Rat FGF-21 Quantikine ELISA Kit, R&D Systems; Mouse albumin and haptoglobin ELISA kit, Abcam). Plasma Albumin and Haptoglobin levels Lipids were extracted from liver tissues with chloroform/methanol mixture (2:1 v/v) and quantitated using Serum Triglyceride Determination Kit (Sigma), as described previously (28 (link)). All assays were performed according to the manufacturer’s instructions. Blood glucose levels were measured using Ascensia Breeze 2 Blood Glucose Monitoring System (Bayer).