Lung tissue was fixed with 4% PFA for at least 48 h, and then dehydrated and embedded in paraffin. The embedded lung tissue was sectioned into slices with a thickness of 4 μm and subsequently affixed onto slides for staining. AT2 cells were fixed with 4% paraformaldehyde for 30 min at room temperature and then permeabilized with 0.5% Tryon-100 for 30 min. Cells washed with PBS were blocked with 5% bovine serum albumin (BSA) (SW3015, Solarbio, Beijing, China) for 1 h at room temperature.
Immunofluorescence staining was performed using primary antibodies against SPC (1:200, AP53886PU-N, OriGene, Maryland, MD, USA), Ki67 (1:200, GTX00538, Genetex, Texas, USA), or HIF-1α (1:100, AF1009, Affinity, Changzhou, China). Following overnight incubation at 4 °C, the slides were washed and subsequently incubated with the corresponding secondary antibody, IgG Alexa Fluor 594 (1:1000, 8889 s, Cell Signaling Technology, Boston, MA, USA), for 1 h at 37 °C in a light-restricted environment. This was followed by a 5 min incubation with 4′,6-diamidino-2-phenylindole (DAPI, 10 μg/mL) in darkness. Images were promptly captured using a fluorescence microscope.
Immunofluorescence staining was performed using primary antibodies against SPC (1:200, AP53886PU-N, OriGene, Maryland, MD, USA), Ki67 (1:200, GTX00538, Genetex, Texas, USA), or HIF-1α (1:100, AF1009, Affinity, Changzhou, China). Following overnight incubation at 4 °C, the slides were washed and subsequently incubated with the corresponding secondary antibody, IgG Alexa Fluor 594 (1:1000, 8889 s, Cell Signaling Technology, Boston, MA, USA), for 1 h at 37 °C in a light-restricted environment. This was followed by a 5 min incubation with 4′,6-diamidino-2-phenylindole (DAPI, 10 μg/mL) in darkness. Images were promptly captured using a fluorescence microscope.