Prussian blue stain kit

Fresh pig skin was purchased from Jinluo Foods Co., Ltd. (Yantai, China) and was stored at −20°C until further experimentation. Pepsin (3,000 U/mg) and trypsin (1,000–1,500 U/mg) were obtained from Shanghai Lanji Technology Development Co., Ltd. (Shanghai, China). Ferrous chloride (molecular weight 126.75 Da) was purchased from Shanghai Biological Technology of Vibration Spectrum Co., Ltd. (Shanghai, China). Ferrous sulfate tables were purchased from Shanxi Lijiu Pharmaceutical Co., Ltd. (Shanxi, China). Ascorbic acid, anhydrous ethanol, and other reagents used in this study were of analytical grade. The Prussian Blue Stain Kit and hematoxylin-eosin/HE staining kit were obtained from Solarbio Technology Co., Ltd. (Beijing, China).

The hearts were harvested after 6-G treatment for 12 weeks, and the control group was sampled at the same time. The excess blood was removed by perfusion with 10% KCl. The tissue was cut into 4-5 μm sections after being fixed with 4% paraformaldehyde and embedded in paraffin. Hematoxylin and eosin (HE) staining was used to assess the cardiomyocyte cross-sectional area and to trace the outline of striated muscle. A Prussian Blue Stain kit (Solarbio, G1424, China) was used to assess the level of iron. Collagen I and collagen III were detected by immunohistochemistry to evaluate interstitial fibrosis. Briefly, antigen unmasking was performed with citrate buffer (0.01 M, pH = 6.0) in the microwave; then, a ready-to-use immunohistochemical hypersensitivity Ultrasensitive™ SP kit (KIT-9710, MXB, China) was used to block nonspecific binding sites, followed by overnight incubation with antibodies against collagen I (1 : 100, Abcam, ab7778) and collagen III (1 : 100, Abcam, ab34710) at 4°C. Sections were washed with PBS 3 times, 10 min each wash, and then incubated with anti-rabbit secondary antibodies from the kit. Finally, staining was visualized with DAB followed by counterstaining with hematoxylin after washing with PBS. Image-Pro Plus 6.0 was used to analyze the photos.