Prussian blue iron stain kit

Ear-tissue samples from all groups were excised and fixed in 4% formalin until use. All samples were embedded at the hedge in optimal cutting temperature (OCT) (OCT compound; Tissue-Tek^®; Sakura Finetek USA, Inc., Torrance, CA, USA) and instantly frozen in liquid nitrogen. Slides were obtained by cutting the ear block with a cryostat at −20°C. For H&E staining, slides were thawed, hydrated, washed, and stained with an H&E staining kit (Sigma-Aldrich Co.). Images were captured with a Nikon Eclipse Ti inverted fluorescence microscope (Nikon, Tokyo, Japan). The slides were stained with Prussian blue iron stain kit (Beijing Solarbio Science and Technology, Co. Ltd) according to manufacturer’s instructions.

The slices were subjected to dewaxing and rehydration. The Prussian blue iron staining procedure was performed following the instructions of the Prussian Blue Iron Stain kit (G1428; Solarbio). The results were measured using the PA53 biological microscope.

Fe³⁺ in corneal tissues was detected by the Prussian Blue Iron Stain Kit (Solarbio, Beijing, China) according to the instructions. In brief, the corneal paraffin sections were incubated with Perls Working Solution at 37°C for 20 minutes. Next, the sections were exposed to an incubation solution, and enhanced working solution at 37°C for 15 minutes, respectively. Finally, the corneal sections were treated with a redyeing solution. Fe³⁺ levels were assessed by determining the percentage of positive area in corneas using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The detection of Fe²⁺ in CSSCs was carried out using a FerroOrange fluorescent probe (Dojindo, Kumamoto, Japan) following the manufacturer's protocol.²⁴ The CSSCs subjected to the treatment were incubated with 250 µM FerroOrange at 37°C for 30 minutes, after which they were immediately observed under a fluorescence microscope.

The dorsal skin was removed from the euthanized rats, washed with PBS, and fixed with 4% formaldehyde for at least 1 day. The skin samples were dehydrated in graded ethanol, cleared with xylene, infiltrated with paraffin wax, and embedded in paraffin. The embedded sample blocks were cut into 5-μm sections and stained with hematoxylin and eosin (H&E) for histological analysis. The sections were then washed in H₂O and tissue iron deposits were detected using the Prussian blue iron stain kit (Solarbio). Signal was enhanced with Fast 3,30–diaminobenzidine (DAB; Sigma, St Louis, MO, USA) for 2 min. The samples were then examined using an optical microscope (X51; Olympus Corporation).

PSC NP-labeled cells were fixed with 4% (wt/vol) paraformaldehyde (Sinopharm, Shanghai, China) for 30 minutes, incubated in iron staining solution (potassium ferrocyanide and hydrochloric acid) for 30 minutes following the manufacturer’s instructions for Prussian blue Iron Stain Kit (Solarbio, Beijing, China; G1424). It was followed by a 1× PBS (Gibco) rinse and Eosin staining. The images were captured by a microscope (Nikon, Japan).