Anti involucrin

To evaluate the expressions of differentiation markers in the epidermis, the mice dorsal skin were obtained at the 84 h, and was subjected to fixation using 10% neutral buffered formalin. This was followed by embedding the samples in paraffin and sliced into 6 μm thick sections. For immunohistochemistry, the sections were incubated in 3% H₂O₂ for 25 min and then blocked by 3% BSA for 4 h. After that, the sections were incubated overnight at 4 °C with the immunohistochemical markers: anti-Filaggrin (1:500; Absin, Shanghai, China), anti-Involucrin (1: 200; Proteintech, Wuhan, China). After washed with PBS three times, the sections were incubated with horseradish peroxidase-conjugated secondary antibodies for 4 h at 37 °C and then reacted with 3,3-diaminobenzidine (DAB). All stained sections were examined by DM3000 microscopy (Leica, Wetzlar, Germany) to assess the histological changes.

Extracts of a skin sample or HaCaT cells were prepared and then centrifuged at 12,000 × g to precipitate the insoluble materials. The supernatant was retained and the concentration of protein in the supernatant was measured by a BCA Protein Assay Kit (Beyotime, Jiangsu, China). A sample of supernatant was resolved by SDS-PAGE using a 12% gel. The protein bands in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% skim milk, followed by overnight incubation with anti-Filaggrin, anti-Involucrin, anti-PPARα, anti-PPARγ, anti-LXR (Proteintech, Chicago, IL, United States), or anti-ABCA1 (Absin, Shanghai, China). After that, the blot was washed and incubated with the appropriate secondary antibody (Cell Signaling Technology, Beverly, MA, United States). Finally, the blot was subjected to a detection assay using a chemiluminescence substrate (Pierce, Rockford, IL, United States), and images of the blot were acquired using an Amersham Imager (GE Healthcare Biosciences, Pittsburgh, PA, United States).