2.3. Skin immunohistochemistry analysis

Skin sections were prepared as previously described. 7 Briefly, 5‐μm‐thick sections of 10% neutral formalin‐fixed, paraffin‐embedded tissues were cut on silane‐coated glass slides, de‐paraffinized three times with xylene and then dehydrated through a graded alcohol bath. Subsequently, the sections were washed with PBS before immunostaining and pretreated by blocking with 1% BSA for 30 minutes to prevent nonspecific binding of the antibodies. The sections were then incubated overnight with the primary anti‐antibody (1:500 dilution) in PBS containing 0.3% Triton X‐100 and normal goat serum, and subsequently with the secondary antibody (1:100 dilution) for 60 minutes, followed by incubation in ABC solution for 1 hour at room temperature. Colour development was performed by incubating the sections with streptavidin for 40 minutes. The slides were incubated for 10 minutes with 50 μg/mL 4′,6‐diamidino‐2‐phenylindole (DAPI). Images were viewed using a microscope (Olympus Microscope System BX53; Olympus, Tokyo, Japan). The results were quantified by measuring the fluorescent density at 40× magnification using ImageJ software (Bethesda, MD, USA); data are presented as a percentage of the pH 7.40 group values.