Hematoxylin eosin

Three-dimensional human oral epithelial tissue models with the stratum corneum (SkinEthic HEG; Episkin, Lyon, France) were cultured according to the manufacturer’s instructions. Each insert dish with the tissue was transferred to a 24-well plate previously filled with 300 μL of maintenance medium. NE was added to an insert dish at a concentration of 100 mU/mL, followed by incubation at 37 °C in 95% air and 5% CO₂ for 12 h. Thereafter, the tissue models were carefully removed from the insert dish together with the polycarbonate filter. The tissue models were then embedded in an optimal cutting temperature compound (Sakura Finetek Japan Co., Ltd.) and snap-frozen in liquid nitrogen, and frozen sections (20 μm) were prepared in a cryostat. These sections were stained with hematoxylin–eosin (Sakura Finetek) and imaged using a BIOREVO BZ-9000 microscope (KEYENCE).

Fresh liver biopsies from rats were fixed in 2% p-formaldehyde in 0.1 M phosphate buffer at pH 7.4 for 24 h and processed routinely until they were embedded in Paraplast wax. Samples were sliced into 8 μm thick sections and stained with hematoxylin–eosin (Sakura Finetek, Mestre, Italy). Slices were then evaluated under a light microscope (Nikon Eclipse E800). Lipid droplet number and area were estimated using ImageJ software version 9.2.0.

Cross-sections (thickness, 10 μm) were cut from frozen samples of 14 days OL plantaris muscle and stained with hematoxylin & eosin (Sakura Finetek) according to standard procedures. Stained cross-sections were observed under a bright field microscope (Eclipse TE300; Nikon, Tokyo, Japan) and captured with a digital camera (DS-5M; Nikon, Tokyo, Japan). From photomicrographs of each muscle, cross-sectional area (CSA) of ≥1000 fibers was measured using ImageJ v. 1.46r (National Institutes of Health, Bethesda, MD, USA). The plantaris muscle has slow fibers in its deep region and fast fibers with different fiber size in its middle to surface region [22 (link)]. To avoid such a region-dependent bias, the measurement of CSA were made evenly for fibers in all captured images. As with muscle weight, fiber CSA is thought to be affected by body weight, so that it was expressed relative to body weight, too (CSA/body weight).