Histology and immunofluorescence

Lungs were fixed in 4% paraformaldehyde (PFA) for 2 days at RT, embedded in paraffin, and sectioned at 3 µm. Haematoxylin and eosin (H&E) staining was used to assess pulmonary pathologies. A 5-grade scoring system was adopted to describe the severity of the lung damage from least severe to most severe as described previously.²⁰ TUNEL assay was used to assess apoptotic cells in lung tissues via a DeadEndTM Fluorometric TUNEL System (Promega, USA).

Paraffin-embedded sections were incubated with 3% H₂O₂ to block endogenous peroxidases and then subjected to an EDTA buffer for antigen retrieval. The sections were then incubated with blocking buffer (5% goat serum). Primary antibodies used for immunohistochemistry or immunofluorescence analysis included rabbit anti-myeloperoxidase (anti-MPO, Abcam, ab9535), rabbit anti-CD4 (Abcam, ab183685), rabbit anti-CD8α (CST, 98941), and rabbit anti CD103 (Abcam, ab224202).