K646 100

Mouse iBAT samples were boiled in 30% KOH for 1 h and placed on ice. Crude glycogen was precipitated in 100% ethanol. After centrifugation, glycogen precipitates were dissolved in 10 μN HCl and reprecipitated in 100% ethanol. The procedure was repeated once to remove residual glucose. Final glycogen precipitates were air-dried and dissolved in distilled H₂O. Glycogen levels were measured using a kit from BioVision (K646-100, Milpitas, CA, USA) based on the manufacturer’s protocol.

Biochemical quantification of glycogen was performed by a commercial glycogen assay kit (K646‐100, BioVision). After microwave or perfusion fixation, the weight of a whole brain or brain tissue specimens was measured before homogenization. Homogenized samples were adjusted to have a concentration of 10 mg/200 µL in distilled water, followed by centrifugation by 13,000 rpm for 5 min. Supernatant and glycogen standard were transferred to a 96 well plate, followed by incubation with 1 µL hydrolysis enzyme mix for 30 min. Subsequently, the samples were incubated with 1 µL development enzyme mix and 0.3 µL OxiRed probe for 30 min in room temperature. The fluorescence intensity of samples was measured by a microplate reader (Thermo Scientific, Varioskan Flash; Ex/Em 535/587nm). After measurement, glycogen concentration was calculated from the calibration curve obtained by the glycogen standard. The final glycogen concentration was computed by subtracting the background value (the signal without hydrolysis enzyme mix).

Glycogen concentration in the frontal cortex and hippocampus was measured with an enzymatic method using a fluorimetric assay kit (#K646-100; BioVision, USA). Samples were homogenized in 5 volumes of redistilled water, boiled for 5 min at 96 °C to inactivate the enzymes and centrifuged at 20,000×g for 20 min. Forty microliters of each supernatant was transferred to a 96-well plate along with blanks and standards (0.2, 0.4, 0.6, 0.8, and 1.0 μg per well). In the selected assay, glycogen is hydrolyzed to glucose (by glucoamylase), which is subsequently oxidized and reacts with an OxiRed probe to produce a fluorophore. Fluorescence intensity was measured at excitation and emission wavelengths of 535 and 590 nm, respectively, with a fluorometer (Tecan Infinite 200 Pro, Switzerland). The concentration of glycogen was calculated by subtracting the background fluorescence (amount of glucose in unhydrolyzed samples) from the fluorescence intensity of the samples after hydrolysis. Glycogen levels were then calculated from the standard curve and displayed as μg/mg of protein.