Epidem epi 200

The human epidermal 3-D model (EpiDem EPI-200; MatTek, Ashland, MA, USA) was treated with or without Gly or Th2 cytokines (IL-4 [60 ng/mL] and IL-13 [60 ng/mL]) for 6 days. Culture medium and reagents were replaced every 2 days. The skin samples were fixed in Super Fix (Kurabo, Osaka, Japan) overnight, embedded in paraffin by the conventional method and cut into 3-μm thick sections. Antigen retrieval was performed using Heat Processor Solution pH6 (Nichirei Biosciences, Tokyo, Japan) at 100°C for 40 min, and endogenous peroxidase was blocked by incubating the sections with 3% H₂O₂ (Nichirei Biosciences). The sections were then incubated with anti-filaggrin (1:1000) antibody at 4°C overnight, followed by incubation with secondary antibody, N-Histofine Simple Stain MAX-PO MULTI (Nichirei Biosciences). Immunodetection was conducted with 3,3-diaminobenzidine as chromogen, followed by light counterstaining with hematoxylin. Sections stained without primary antibody served as a negative control.