Hematoxylin and Eosin Staining
Collected back skin tissues from mice were conserved in 10% formaldehyde solution for 72 h, then dehydrated. Hematoxylin and eosin (H&E) staining was performed on 4 mm thick paraffin-embedded sections. The tissue sections were examined under an OLYMPUS light microscope. Histological scores were evaluated by a cumulative score containing four independent scoring criteria (0-4): Epidermal thickness, inflammatory cells infiltrate, capillaries dilatation, and parakeratosis. Three different fields of view were used to measure the thickness of the mice’s epidermis.
Immunohistochemistry
Paraffin sections were deparaffinized with xylene and rehydrated in decreasing ethanol concentrations; then, antigen retrieval was carried out with the EDTA buffer (PH 9.0) for 25 min at 100°C. To neutralize the endogenous peroxidase, sections were cooled down to room temperature and incubated with 3% H2O2 for 10 min to neutralize the endogenous peroxidase. After washing twice with PBST (1× PBS with 0.05% Tween 20), sections were blocked with 10% goat serum for 45 min and incubated in the specific primary antibodies against Ki67, IL-17A, CD3, F4/80, and myeloperoxidase (MPO) with appropriately diluted ratio overnight at 4°C. After washing with PBST, tissues were incubated with HRP-conjugated goat anti-rabbit secondary antibody for 45 min at room temperature. All sections were stained using DAB for a few seconds and lightly counterstained with hematoxylin. Images were obtained with an Olympus BX41 microscope.