Hematoxylin and Eosin (H&E) staining and immunohistochemistry (IHC) assay

The organs (heart, liver, spleen, lung, and kidney) and tumors were collected, dehydrated with graded alcohol, soaked in xylene solution, and then embedded in paraffin. The paraffin tissue sections (5 μm thickness) were dewaxed in xylene, soaked in graded alcohol, and dehydrated. After staining with H&E, the samples were photographed by microscopy (Olympus, Tokyo, Japan).

Deparaffinized and dehydrated paraffin tissue sections (5 μm thickness) were obtained. The cells were treated with 3% hydrogen peroxide at room temperature for 10 min in the dark to block the intrinsic catalase. Then, the sections were subjected to hot antigen retrieval with 10 mM sodium citrate buffer (pH 6.0). Normal serum was used to block nonspecific binding sites at 37 °C for 1 h. Ki67 (Abcam, ab16667) was treated overnight at 4 °C, and the secondary antibody was added at 37 °C for 30 min. Streptavidin-peroxidase complex was added for 30 min. Furthermore, sections were stained and re-stained using DAB and hematoxylin. After washing with running water, the samples were treated with graded alcohol, cleared to xylene, and then mounted for microscopy. IHC images were acquired using a fluorescence microscope system (Olympus, Tokyo, Japan).