Immunohistochemical staining was performed for T-bet (1:500; sc-21763; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) to evaluate Th1 cells; GATA binding protein 3 (GATA3; 1:500; sc-268; Santa Cruz Biotechnology, Inc.) was used for the Th2 cells; forkhead box protein P3 (FOXP3; 1:200; NB100-39002; Novus Biologicals, Centennial, CO, USA) was used for regulatory T (Treg) cells; and RAR-related orphan receptor (ROR)γ (1:1000; ab207082; Abcam, Cambridge, UK) was used for Th17 cells.
The sections were rehydrated, subjected to antigen retrieval using Dako Target Retrieval Solution (pH 9.0; Dako North America, Inc., Carpinteria, CA, USA) for 10 min at 121 °C, blocked for endogenous peroxidase with 0.3% H2O2 in methanol, and incubated for 30 min. After washing with phosphate-buffered saline, the sections were blocked for nonspecific binding using Blocking One Histo (Nacalai Tesque, Inc., Kyoto, Japan) for 15 min at room temperature and then incubated with the primary antibody overnight at 4 °C. The sections were reacted using peroxidase stain 3,3-diaminobenzidine (DAB) kit (Nacalai Tesque, Inc.) for 1 h and developed with DAB solution. Hematoxylin counterstaining was performed following the DAB reaction.
The sections were rehydrated, subjected to antigen retrieval using Dako Target Retrieval Solution (pH 9.0; Dako North America, Inc., Carpinteria, CA, USA) for 10 min at 121 °C, blocked for endogenous peroxidase with 0.3% H2O2 in methanol, and incubated for 30 min. After washing with phosphate-buffered saline, the sections were blocked for nonspecific binding using Blocking One Histo (Nacalai Tesque, Inc., Kyoto, Japan) for 15 min at room temperature and then incubated with the primary antibody overnight at 4 °C. The sections were reacted using peroxidase stain 3,3-diaminobenzidine (DAB) kit (Nacalai Tesque, Inc.) for 1 h and developed with DAB solution. Hematoxylin counterstaining was performed following the DAB reaction.