2.4. Tissue preparation and H& E morphology staining for adipose

Adipose tissue sections were fixed in a 1X zinc formalin fixative (Sigma Aldrich, St. Louis, MO) for 12 h, and then rehydrated in sucrose buffer (30 % in PBS, w/v) overnight. Tissue was then embedded in 7.5 % gelatin/1X PBS, solidified on a dry ice block, sectioned at 10 μm on a cryostat, placed on Poly-L Lysine slides (Thermo Fisher Scientific, Waltham, MA), and then processed through H&E, Nile Red, or immunofluorescent staining. Remaining tissue samples/slides were stored at -80 °C. For H&E staining of adipose, the slides were air-dried at RT for 15 min, fixed in ice-cold 1:1 methanol:acetone solution for 30 min, and rinsed 2x for 5 min each in 1X PBS. Slides were then stained with hematoxylin (1 min RT), rinsed in PBS, and then counterstained with eosin (30 s at RT), rinsed in PBS, cover-slipped, and imaged on an EVOS XL Core (Thermo Fisher Scientific) at 20x and 40x for analysis. A blinded participant measured the adipocytes. 40x images were used, and a minimum of 4–15 adipocytes were measured on each section, 8–10 sections from each animal, for a total of at least 100 cells per animal, and n = 4–5 animals per group, using Image J software (NIH). All adipocytes were measured along the longest axis in the view/plane for each measurement.