Histological Assessment of Adipocyte Size in Visceral Fat

To measure the size of adipocytes in visceral fat, the adipose tissue slides were stained with hematoxylin & eosin. The adipose tissue slides were deparaffinized with xylene and then rehydrated using progressive ethanol solution (100%, 90%, 80%, and 70%) for 1 minute each step. After rinsing the slides with running water, the slides were immersed in hematoxylin solution and washed with tap water for 3 minutes. The tissue slides were immersed into the eosin solution for 1 minute and washed with running water. Cover slides were mounted using xylene-based DPX solution (Sigma-Aldrich, MO, USA) and visualized with an optical microscope (BX53M; Olympus, Japan). The adipocytes size of visceral fat tissue was determined by randomly capturing 10 visceral fat images and the adipocyte area was measured from the cross-sectional area of the adipocyte membrane by the Image J software (NIH, DC, USA) [28 (link), 29 (link)].