2.7. Haematoxylin–Eosin (HE) Staining

The histopathology of the lung was assessed by performing HE staining [22]. After the rats were sacrificed, the lung was quickly harvested on ice and fixed in 4% paraformaldehyde, dehydrated, cleared in xylene and embedded in paraffin. Six-mm-thick lung sections were prepared for HE staining and observed by microscopy (Olympus, Tokyo, Japan). For semiquantitative analysis, a total of 2 images from the lung in each rat were recorded (3 rats per group). Each image was scored for inflammation and macrophage accumulation in alveoli using a semiquantitative scale of 0 (no pathologic changes) to 4 (obvious pathology approaching maximal). Semiquantitative analysis of each lesion was scored by the presence of individual affected foci (score 1), increased frequency and size of individual lesions with occasional confluence (score 2), large areas of involvement and confluence (score 3), or complete lung involvement with the lesion in question (score 4) [23].