We obtained dorsal skin samples on the final day of examination. The skin samples were fixed in phosphate-buffered paraformaldehyde (4%), embedded in frozen Tissue-Tek, an OCT compound, and cut into 5 µm sections. The sections were stained with hematoxylin-eosin (HE) in accordance with the established procedures to enable histological analysis of the skin. To evaluate collagen expression, samples were stained by the Masson trichrome technique (trichrome stain kit [modified Masson's]; ScyTec Laboratories, Inc., Logan, UT, USA) 23. Additionally, skin specimens were stained with toluidine blue to visualize mast cells, and the stained skin tissues were microscopically evaluated following conventional procedures. Thereafter, the specimens were stained using an antibody for immunohistological analysis, as described previously 22. The skin specimens were incubated with a rabbit polyclonal anti-collagen IV (1:1000; Abcam) primary antibody. The specimens were subsequently incubated with fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody (1:30; Dako Cytomation, Glostrup, Denmark). The expression of collagen IV was evaluated immunohistochemically by fluorescence microscopy.

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