The excised skin of mice was fixed in 10% formalin and embedded in paraffin. For histopathological examination, the tissue slices of 5-μm thickness were stained with hematoxylin and eosin (H&E) under the microscope (Olympus BX61, Tokyo, Japan). Epidermal thickness was calculated based on the means of four random sites of view per tissue and quantitated by ImageJ software as previously described (Liu X. et al., 2017 (link)). For immunohistochemistry, the tissue slices of 5-μm thickness were deparaffinized and incubated in hot citric acid buffer (PH 6.0) for antigen retrieval. After immediate cooling, slides were blocked with 5% BSA in TBST for 1 h and incubated with primary antibodies against ANGPTL4 (A2011, ABclonal) and Ki67 (GB13030-2, Servicebio) at 4°C overnight, followed by secondary antibodies at 1:200 for 30 min at room temperature. The immunostaining was visualized by 3,3-diaminobenzidine (Vector Laboratories, Burlington, CA, United States). Quantification of the DAB intensity in the images was performed using ImageJ software.
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