Transmission Electron Microscopy

Samples used for TEM were prepared, as described previously (17). The harvested skin sample was cut into small pieces (5 mm³) and fixed in 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, United States) in 0.1 M PBS for 24 h at 4°C. The samples were then post-fixed with 1% osmium tetroxide solution for 12 h, dehydrated with a descending ethanol series for 2 h, and embedded in epon resin. The area representative of the dermis was retained for ultrastructural analysis. Subsequently, 80-nm-thick ultrathin sections were cut and placed on copper grids for observation under a JEM-1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan).