The paraffin-embedded lung sections were dewaxed and cleared with xylene, followed by hydration with gradient ethanol. Then, the sections were retrieved with sodium citrate solution (pH 8.0; Beyotime, Shanghai, China) for 20 min. The sections were blocked with blocking solution (7.5% goat serum and 0.5% Triton X-100 in PBS; Beyotime, Shanghai, China) for 1 h and incubated with primary rabbit (anti-CD31, Abcam, ab6994, 1:200) anti-Sema7a (Abcam, ab23578, 1:200), anti-RAGE (Abcam, ab21171, 1:200), and anti-Ki67 (Abcam, ab15580, 1:1000) antibodies overnight at 4 °C. The sections were washed twice with PBST (PBS with 1% tween) and once with PBS, and then incubated with secondary antibodies and DAPI mix for 1 h at room temperature. After washing for three times with PBS, the sections were covered with anti-florescence quencher and sealed with nail polish.
TUNEL staining was applied using the one-step TUNEL apoptosis assay kit according to the manufacturer's instructions. Briefly, the sections were dewaxed, rehydrated, and retrieved using antigen. Then, the sections were incubated with 20 μg/mL of protein K for 30 min at room temperature. After washing with PBS, the sections were incubated with 50 µL of TUNEL detection solution (5 µL of TdT enzyme and 45 µL of fluorescein labeling solution) at 37 °C for 1 h.
TUNEL staining was applied using the one-step TUNEL apoptosis assay kit according to the manufacturer's instructions. Briefly, the sections were dewaxed, rehydrated, and retrieved using antigen. Then, the sections were incubated with 20 μg/mL of protein K for 30 min at room temperature. After washing with PBS, the sections were incubated with 50 µL of TUNEL detection solution (5 µL of TdT enzyme and 45 µL of fluorescein labeling solution) at 37 °C for 1 h.