Prussian blue

Briefly, the aorta tissue sections isolated from mice were first deparaffinized and rehydrated. Perls’ blue staining was performed as previously described (39 (link)). For Perls’ blue staining, the sections were stained with a 1:1 mixture of Prussian blue (catalog no.: G1424; Solarbio) staining solution A and solution B for 30 min, washed twice with distilled water, stained with Prussian blue staining solution C for 3 min, and washed with running water. They were dehydrated with three changes of 100% ethanol and two changes of xylene for 5 min each and sealed with neutral balsam. Images were obtained using a positive fluorescence microscope (BX63; Olympus).

After measuring cardiac function, the rats were anaesthetised and killed by cervical dislocation. Myocardial tissues were resected, fixed in 10% formaldehyde, embedded in paraffin, and sliced into 5 μm sections. After dewaxing and rehydration, the sections were stained with haematoxylin–eosin (HE) (Beyotime, Beijing, China) to determine pathological damage, MASSON (Haematoxylin, Masson's ponceau acid fuchsin solution, and aniline blue; Solarbio, Beijing, China) to determine fibrosis, Prussian blue (Solarbio) to determine iron deposition, or terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL; Beyotime) to determine apoptosis. A part of each section was used to detect UL3 expression by immunohistochemistry (IHC). Stained sections were observed under a microscope (Olympus, Tokyo, Japan).

Three similar frozen brain sections were collected per rat, and Prussian blue staining was performed per the manufacturer's instructions (Solarbio) and used to determine ferric iron deposition on day 10. Briefly, sections were rinsed three times in distilled water and incubated in Prussian blue solution (1% potassium ferrocyanide and 1% HCl [v/v]) for 20 min. Sections were then rinsed with distilled water three times and dehydrated through a series of ethanol gradients. Dimethylbenzene was used to transparentize the tissue, a neutral gum seal sheet was used, and the tissues were air‐dried. The dried samples were viewed microscopically, and images were collected for analysis.