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.