Histological Assessment of Skin Changes

For histologic evaluation of epidermal thickness, LI, and the number of dermal infiltrated inflammatory cells, groups of mice (n = 4 mice/group) were treated with the triterpene compounds according to the short-term protocol regimen. BrdU (100 μg/g B.W.) dissolved in PBS was injected i.p. to mice 30 min prior to sacrifice. Forty-eight hrs after the last TPA treatment, dorsal skin was excised and fixed in 10% neutral-buffered formalin. The fixed skin samples were embedded in paraffin, sectioned (4 μm) and stained with toluidine blue O (Fisher Scientific, Pittsburgh, PA), anti-BrdU (Abcam, Cambridge, MA), or anti-CD3 (Cell Signaling Technology, Beverly, MA). Epidermal thickness and LI were determined as described previously [62 (link)]. The number of inflammatory cells in the dermis was counted per 200 mm² field as previously described [59 (link)].