Nile Red Staining for Lipid Quantification

Nile Red staining of tissue sections was performed similar to the method previously described^{57 (link)}, with our experimental details listed below. The stock solution was prepared by dissolving Nile Red (Sigma 72485) first in acetone at 500 μg/mL then diluting to a working solution of 2.5ug/mL. Skin tissue sections were fixed in 10% formalin for 5 min, rinsed briefly in PBS, then incubated in the working solution for 10 min at room temperature. The slides were subsequently washed with PBS and stained by NucBlue Fixed Cell ReadyProbes Reagent (DAPI, Invitrogen) for 5 min, and were mounted with anti-Fade Fluorescence Mounting Medium (Abcam). For quantification, the thickness of Nile-Red-positive regions in each image was measured at 3 regions using the Image J software (NIH). The average thickness from 3 measures of each image were calculated, and a total of 26 images per condition were included for statistical analysis (T-test) using Prism.