Prussian blue iron stain kit with eosin

Iron staining was performed using the Prussian Blue Iron Stain kit (With Eosin) (G1424, Solarbio). The kit consisted of A1: Perls staining solution A and A2: Perls staining solution B. After routine dewaxing and rehydration of liver paraffin sections, A1 and A2 were mixed in equal amounts, and the sections were stained with Perls working solution dropwise for 25 min, washed with PBS for 3 min, washed twice, and then the background was lightly stained with eosin staining solution for 15 ~ 30 s, and finally rinsed with tap water for 3 s. Finally, the sections were dehydrated, transparent, and sealed. The nuclei as well as other tissues were stained red, while iron-containing heme or trivalent iron was stained blue. The staining results were quantitatively analyzed using ImageJ software.

After referring to the Prussian Blue Iron Stain Kit (with eosin) (Solarbio, Beijing, China) protocol, paraffin sections of ovarian tissues (5 μm) were baked at 60 °C for 1 h, deparaffinized in dimethylbenzene and graded alcohol to water. The sections were further immersed in staining reagent Perls stain A for 10 min, then rinsed thoroughly in distilled water and immersed in eosin staining solution for 30 s. Slides were finally dehydrated in graded alcohol, cleared in dimethylbenzene, and mounted in neutral resin for microscopic examination.