Lung and Heart Tissue Harvesting and Analysis

Both the RA control and BPD mice were anesthetized (using an overdose of a cocktail of xylazine-ketamine) and lung and heart tissues were harvested after perfusion and fixed overnight in 4% paraformaldehyde. Fixed tissues were then washed in fresh PBS, and stored in 70% ethanol before being processed in an autostainer (Ventana, CA, USA) by the Histology Core Facility of Wistar Institute, Philadelphia to be stained with hematoxylin and eosin (H&E) for lung morphometry or immunohistochemistry/immunofluorescence as previously described [19 (link),23 (link),24 (link)]. Immunofluorescence staining was performed for Ki67 (Abcam, MA, USA 1:10) SP-C (Abcam, MA, USA, 1:100), RAGE (R&D Systems, Minneapolis, MN, USA, 1:100), and Von Wilebrand Factor (vWF-DAKO, 1:100) on lung paraffin sections following the protocol as described earlier [24 (link)], while TUNEL staining was performed following the manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN, USA).