White Adipose Tissue Histological Analysis

After euthanasia, the mesenteric (WAT-M) and inguinal (WAT-I) WAT depots were excised, weighed, and a fragment was immediately transferred to Alfac, a histological fixation solution constituted by a mixture of alcohol (80%), formol (10%), and glacial acetic acid (5%), during 24 h. After this period, the WAT tissue samples were transferred to an alcoholic (70%) solution for histological procedures. For this, WAT depots were diaphanized in xylol, dehydrated in alcoholic solution and embedded in paraplast (McCormickTM; Leica Microsystems Pty Ltd., Sydney, Australia), being finally submitted to the microtomy procedures. Semi-serial cuts (5 μm) were performed and stained with hematoxylin and eosin (H&E). Tissues from five to six rats per group were used to assemble the slides for histology (three slides per rat, containing at leastthree slices each). Images of the slides were captured using a photomicroscope (Olympus BX60, Tokyo, Japan) at a magnification of 40x. Adipocytes, size (μm²), and number (number/field) were measured using an image analysis system (Image J 1.39f, NIH–Bethesda, MD, United States). A total of 50 adipocytes were analyzed per section.