Xylene

Hematoxylin and eosin (H&E) staining was performed as previously described (15 (link),16 (link)). To deparaffinize the paraffin sections, slides were submerged for 5 min in a slide jar filled with xylene (Sinopharm Chemical Reagent Co., Ltd.), repeating this procedure twice and using fresh xylene each time. Subsequently, sections were rehydrated by running slides through a series of decreasing concentrations of EtOH (Sinopharm Chemical Reagent Co., Ltd.). Hematoxylin (Shanghai Jingke Chemical Technology Co., Ltd.) was used to stain the tissues for 5 min at room temperature, and after washing with tap water, differentiation in staining was visualized by submerging for 30 sec in 1% acetic acid. To visualize the blue staining, the sections were rinsed under running tap water for 5 min, after which the slices were dyed in eosin solution (Shanghai Jingke Chemical Technology Co., Ltd.) for 2 min at room temperature. After dehydration with EtOH and transparency with xylene, the slices were sealed with neutral balsam (G8590-100; Solarbio). Finally, tissues were imaged under an electron microscope (400x magnification; OLYMPUS CX-21; Shanghai Xinyu Biotechnology Co., Ltd.).

Paraffin sections were incubated with Xylene (Solarbio) for 25 min for dewaxing. They were then hydrated with alcohol, eluted with PBS and placed in sodium citrate buffer (Solarbio) for antigen repair. The sections were blocked with goat serum (Solarbio) and incubated with primary antibodies of ki‐67 (ab15580, 1:1000, Abcam) and ASCT2 (ab237704, 1:1600, Abcam) at room temperature for 1 h. Then, DBA color developer (Solarbio) and hematoxylin (Solarbio) were added to these sections for 2 min. After that, the slices were sealed by dehydration and observed under a microscope.