Dissected liver and tumor tissue were fixed in 4% formaldehyde for 1 day and embedded in paraffin (both Sigma‐Aldrich). Next, the tissue was cut into 0.5 mm3 sections by a rotary microtome (Leica Microsystems GmbH, Wetzlar, Germany). Serial sections were cut from the paraffin‐embedded tissues at a thickness of 5 µm for H&E, the Prussian blue, immunohistochemical, immunofluorescence staining, and TUNEL. H&E staining was done with conventional methods using Harris' hematoxylin (Servicebio, Shanghai, China).
Prussian blue staining: Prussian blue staining was performed to detect intracellular iron. Briefly, sections were stained with 1:1 mixture of potassium ferrocyanide (Sigma‐Aldrich, USA) and hydrochloric acid. After being washed with distilled water, slides were then counterstained with nuclear fast red solution and washed with distilled water. Finally, the stained sections were finally dehydrated in 95% ethanol, and finally in 100% ethanol.
Prussian blue staining: Prussian blue staining was performed to detect intracellular iron. Briefly, sections were stained with 1:1 mixture of potassium ferrocyanide (Sigma‐Aldrich, USA) and hydrochloric acid. After being washed with distilled water, slides were then counterstained with nuclear fast red solution and washed with distilled water. Finally, the stained sections were finally dehydrated in 95% ethanol, and finally in 100% ethanol.