Hematoxylin and Eosin Staining Protocol

H&E staining was performed as previously described (Kharkovets et al., 2006 (link); Frisina et al., 2016 (link)). Slides were placed in a series of clearing xylene solutions for 30 min and rehydrated in a graded series of ethanol (100%, 100%, 95%, 80%, 70%, and 50%) and briefly washed in distilled water. The slides were then incubated with hematoxylin (Service bio, China) for 5 min, and washed with running tap water to remove excess hematoxylin followed by differentiation in 1% acid alcohol for 10 s and washed with running tap water for 1 min. This step was followed by an incubation in the eosin counterstain, subsequent dehydration in a graded series of ethanol (80%, 95%, 95%, 95%, 100%, and 100%), and immersion in xylene. At last, the slides were cover-slipped with resinous mounting medium.