IL-31 expression in paraffin-embedded skin tissues was assessed by immunohistochemistry (IHC)[16 (link)]. Briefly, the prepared glass slides were deparaffinized by incubating in xylene solution, followed by hydration using serial alcohols, antigen retrieval using 1× citrate buffer (Sigma-Aldrich), and blocking using 2.5% normal horse serum (Vector Labs, USA). After washing with PBS, the slides were incubated overnight with ionized calcium-binding adaptor molecule 1 (IBA1) (Fujifilm Wako Chemicals, USA) and IL-31 receptor antibody (Santa Cruz Biotechnology) containing 2.5% normal horse serum and then incubated with HRP-conjugated horse anti-rabbit secondary antibody (Vector Labs) for 2 hours. After treatment with 3,3'-diaminobenzidine solution (Vector Labs), the slides were counterstained using hematoxylin, dehydrated in serial alcohols, and mounted using a xylene-based solution.