Immunofluorescent staining was performed as previously described 14 (link). Primary antibodies used are: mouse anti-GFP (Santa Cruz, CA); mouse anti-ACTA2 (Sigma); rabbit anti-ACTA2 (Abcam, MA); rabbit anti-PDGFRα (Cell Signaling). Nuclei were counterstained with Dapi. For quantitative analyses, multiple images (n>8, each contains 300 to 600 cells) were used to count the ratio of labeled cells. Quantitative data are presented as average values+/-standard error of the mean. Sections from at least three lungs were analyzed for each data point.
For Oil red O staining, frozen sections of neonatal lungs (8 um) were air dried, washed with tap water, rinsed with 60% isopropanol and then stained with freshly prepared Oil Red O solution for 15 minutes. The stained sections were rinsed with 60% isopropanol followed by distilled water and then preserved in VECTASHELD mounting medium with Dapi (to visualize nuclei).
For Oil red O staining, frozen sections of neonatal lungs (8 um) were air dried, washed with tap water, rinsed with 60% isopropanol and then stained with freshly prepared Oil Red O solution for 15 minutes. The stained sections were rinsed with 60% isopropanol followed by distilled water and then preserved in VECTASHELD mounting medium with Dapi (to visualize nuclei).