Mice were euthanized by intraperitoneal injection of a solution made of ketamin, dormitor, heparin, and saline. After sternotomy, the lung was perfused transcardially (through the right ventricle) by using PBS (1×), then isolated and incubated for 30 min at 4 °C in COLD medium, stored at − 20 °C overnight, and finally stored at − 80 °C till further tissue processing.
For histology, the lung was flushed from the right ventricle to remove blood cells then perfused through the trachea with a pressure of 20 cm H2O with 5 ml 4% PFA. The trachea was tied off with a string, and the lung was removed and placed in 4% PFA for max. 24 h at 4 °C. Lungs were then progressively dehydrated (30, 50, 70, and 99% ethanol, 3 h each), incubated in xylole, then in paraffine overnight and finally embedded with a Leica embedding machine (EG 1150C). Paraffin blocks were kept cold, and 5 μm sections were cutted. Hematoxylin and eosin staining was performed according to protocols previously published.
For histology, the lung was flushed from the right ventricle to remove blood cells then perfused through the trachea with a pressure of 20 cm H2O with 5 ml 4% PFA. The trachea was tied off with a string, and the lung was removed and placed in 4% PFA for max. 24 h at 4 °C. Lungs were then progressively dehydrated (30, 50, 70, and 99% ethanol, 3 h each), incubated in xylole, then in paraffine overnight and finally embedded with a Leica embedding machine (EG 1150C). Paraffin blocks were kept cold, and 5 μm sections were cutted. Hematoxylin and eosin staining was performed according to protocols previously published.