Necropsy and Tissue Sampling

Sheep were euthanized by IV overdose of barbiturate and then lung tissues were dissected out. The targeted individual segments were cut and inflated with a 1:1 mixture of sterile PBS: optimal cutting temperature compound solution by pumping this solution through the bronchi. This technique maintains lung tissue in inflated condition before fixation for paraffin processing and prior to freezing for cryo-sectioning. Several 0.5 cm thick transverse lung tissue blocks were cut. Slices from the most caudal part of all differentially treated lung lobes were fixed in 4% neutral-buffered formalin and processed in paraffin for the assessment of histopathology. The rest of the lung tissue blocks were embedded in optimal cutting temperature compound and snap-frozen in liquid nitrogen. These lung samples were then kept at −80°C until required for further analysis.