10 mg of brown adipose tissue (BAT) samples were homogenized with a dounce homogenizer and 200 μL RIPA buffer. Samples were centrifuged at 4 °C and pelleted down. Supernatants were centrifuged again at 4 °C, lysates were frozen at −80 °C. Lysates were diluted 2× and run with a 4-hydroxynonenal (4-HNE) assay kit (ab238538, Abcam, Cambridge, MA) on an Epoch microplate reader (BioTek Inc., Winooski, VT) at an absorbance of 450 nm in duplicates and results were plotted along a standard curve. Lysate protein concentration was calculated with a Pierce BCA Protein kit (ThermoScientific, Rockford, Il). Lysates were diluted by 4× and 2% SDS was added to samples to limit interference by lipids (Kessler and Fanestil 1986 (link)). Samples were measured on an Epoch microplate reader (BioTek Inc., Winooski, VT) at an absorbance of 562 nm in duplicates and results were plotted along a standard curve. We selected BAT to measure 4-HNE due to its high metabolic activity in hibernation. Recent data may indicate metabolically active tissues such as BAT might have higher levels of molecular damage over hibernation in AGS (Wilbur et al. 2019 (link)) and our lab has shown BAT is the only tissue to show oxidative stress after arousal (Orr et al. 2009 (link)).