A602440 0050

Antigen retrieval was performed in the lung slices after dewaxing and rehydration, followed by incubation with 3% H₂O₂ (10011218, Sinoreagent, Shanghai, China). The samples were blocked in 1% bovine serum albumin (BSA; A602440-0050, Sangon, Shanghai, China) for 15 min. The sections were incubated with ST2 antibody (1:50; 11920-1-AP, ProteinTech, Wuhan, China) at 4°C overnight and subjected to HRP-labeled goat anti-rabbit IgG (1:500; # 31460, ThermoFisher Scientific, USA) at 37°C for 1 h. Additionally, IL-25R expression was detected in the same way. The sections were incubated with IL-25R antibody (1:100; PA5-47051, ThermoFisher Scientific, USA) at 4°C overnight and next incubated with HRP-labeled donkey anti-goat IgG (1:500; ab6885, Abcam, Cambridge, UK) at 37°C for 1 h. Subsequently, these pieces were stained with 3, 3-diaminobenzidine tetrahydrochloride (DAB; DAB-1031, Maixin, Fuzhou, China) and counterstained with hematoxylin (H8070, Solarbio, Beijing, China). The results were observed under a microscope (Olympus, Tokyo, Japan) at 400 × magnification.

After deparaffinization and hydration, tissue sections (5 μm) were treated in antigen retrieval solution. Sections were then treated with 3% H₂O₂ (10011218, Sinopharm Chemical Reagent Co., Ltd., China) for 15 min to block endogenous peroxidase activity, and then incubated with 1% BSA (A602440-0050, Sangon Biotech, China) to block nonspecific antibody binding. Primary antibody was added to the sections at 4 °C overnight, followed by HRP-labeled goat anti-rabbit IgG (1:500, #31460, ThermoFisher, USA) at 37 °C for 1 h. Sections were then treated with DAB (DAB-1031, Maixin-Bio, China) and hematoxylin (H8070, Solarbio, China). Images were taken under a microscope (BX53, OLYMPUS, Japan). Antibody information: NREP (1:100, DF14359, Affinity, China), Ki67 (1:100, AF0198, Affinity, China), CD31 (1:100, A4900, Abclonal, China).

Cells were homogenized in cell lysis buffer (10% sodium dodecyl sulfate (SDS) and 50 mM Tris.HCl (pH 6.8) on ice for 10 min. The tissue samples were sonicated (70 Hz, 90 s) in cell lysis buffer, followed by an additional incubation on ice for 20 min. Then, the lysates were centrifuged at 4 °C for 15 min at 12,000 rpm. The protein denaturation was accomplished by incubation at 100 °C. Equal amounts (30 µg) of protein samples were separated by 10% SDS-PAGE gels, separated by electrophoresis, and subsequently transferred onto PVDF membranes. After being blocked with 5% skim milk, the membranes were incubated with the primary antibodies, which were diluted with 5% bovine serum albumin (BSA) (Sangon Biotech, A602440-0050, Shanghai, China) at 4 °C overnight, and then appropriate secondary HRP-conjugated antibodies at room temperature 1 h. Antibodies are shown in Table 2.