Ab207082

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.

Paraffin sections of the uterine tissues were boiled in citrate buffer (10 mM sodium citrate, 10 mM citric acid, pH 6.0) in a microwave oven at 92–98°C for 15 min for antigen retrieval. The slides were then blocked with 5% BSA for 30 min at room temperature (25°C). Subsequently, the tissues were incubated with the following primary antibodies: anti-Foxp3 monoclonal antibody (Abcam, Cambridge, MA, ab20034; 1:200) and anti-RORγt monoclonal antibody (Abcam, ab207082, 1:200) overnight at 4°C. The tissues were then incubated with fluorescein-conjugated goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies for 1 h at 4°C in the dark. Subsequently, the nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI, G1012; Xavier Biotechnology Co., Ltd., China) for 10 min. The slides were washed with PBS and mounted with an anti-fluorescence quenching agent. Optical microscopy (Olympus IX73; Tokyo, Japan) was used to capture images.

Total protein from mouse ilea was prepared using a total protein extraction kit (Applygen, Beijing, China) according to the manufacturer's instructions. Protein samples were resolved by SDS-PAGE on pre-cast 4–15% gels (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA) and incubated overnight at 4 °C with rabbit polyclonal antibodies against Foxp3 (ab10901), RORγt (ab207082), STAT3 (ab68153), p-STAT3 (phospho S727, ab30647), STAT5 (ab16276), p-STAT5 (phospho Y694, ab32364), and β-actin (ab179467, Abcam, Cambridge, UK). Horseradish peroxidase-conjugated anti-rabbit polyclonal antibodies (Goat anti-rabbit IgG-HRP, ab6721, Abcam) were used as secondary antibodies and detected using enhanced chemiluminescence (ECL) substrate (Bio-Rad). Band densitometry was performed using Image Lab software (Bio-Rad). The relative index was represented as the ratio of the selected protein/β-actin, and was the average of three biological replicates.