Ma0192

The harvested lung tissues were immediately fixed in 4% paraformaldehyde (Meilunbio, MA0192, Dalian, China) overnight, then dehydrated and embedded in paraffin. Sections (4 μm) were stained with H&E (Novland, IH-017 and IH-018, Shanghai, China) or a modified Masson’s trichrome staining kit (Solarbio, G1345, Beijing, China). Images were obtained under the microscope.

The transwell chambers (Corning, 3422, Corning, NY, USA) were coated with Matrigel and placed in 24-well plates to incubate overnight with culture medium. The treated cells were resuspended in FBS-free medium, and 2 × 10⁴ cells/well were seeded into the upper chamber. A medium containing 10% FBS was then added to the lower chamber and incubated for 24 h. After removal of non-migrating cells, the chambers were fixed in 4% paraformaldehyde (MA0192, Meilunbio, Dalian, China) for 30 min and stained with 1% crystal violet (MA0148, Meilunbio, Dalian, China) for 30 min, and images were acquired using a microscope.

Rat adenohypophysis tissues were fixed in 4% paraformaldehyde (MA0192, Meilunbio, Dalian, China) for 48 h and then made into paraffin sections. After dewaxing and rehydration, the sections were antigenically repaired using EDTA antigen retrieval solution (P0084, Beyotime, Shanghai, China). The samples were sequentially incubated in blocking solution (incubated for 30 min at room temperature), primary antibody (FTO antibody 1:2000 dilution, FOXP2 antibody 1:200 dilution, overnight at 4 °C), and secondary antibody (1:500 dilution, incubated for 1 h at room temperature away from light). The antibodies used were as follows: FTO antibody (ab280081, Abcam, UK); FOXP2 antibody (20,529–1-AP, Proteintech, USA); and anti-rabbit IgG (H + L) antibody (5220–0336, SeraCare, USA). Both antibody incubations were followed by washing three times with PBS (5 min at room temperature). The nucleus was restained with hematoxylin staining solution (C0107, Beyotime, Shanghai, China) after development using a DAB horseradish peroxidase color development kit (P0203, Beyotime, Shanghai, China). The sections were dehydrated and sealed. Images were visualized and collected using an Olympus fluorescence microscope. Three randomly selected images from the immunohistochemistry results were analyzed for positive reaction areas using ImageJ for statistical analysis.