Prussian blue iron staining kit

Briefly, HUMSCs were obtained after normal deliveries following 38–40 week gestations. HUMSCs were cultured in Dulbecco’s modified Eagle medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco) and negative for mycoplasma contamination. All experiments were conducted using HUMSCs at passages 5–8. After reaching 80% confluency, various concentrations of MIONs@PDA (0, 10, 25, 50, 100, and 200 μg/mL) were added to the medium for 12 h. The Cell Counting Kit-8 (Sigma-Aldrich, St. Louis, MO, USA) assay was used to detect the cytotoxicity of MIONs@PDA. HUMSCs were stained with the Prussian blue iron staining kit (Solarbio, Beijing, China), according to the manufacturer’s instructions. The phenotype of HUMSCs was confirmed by the expression of surface markers (positive for CD44-fluorescein isothiocyanate (FITC) and CD105-phycoerythrin (PE), and negative for CD45-FITC) (all from BD Biosciences, San Jose, CA, USA) using a FACSC anto II flow cytometer (FC500; Beckman Coulter Brea, CA, USA), and the data were analyzed with the CXP software (Beckman Coulter). Differentiation capacity of the HUMSCs was assessed by inducing osteocyte and adipocyte differentiation using the StemPro Osteogenesis/Adipogenesis Differentiation Kit (Invitrogen, Waltham, MA, USA) and evaluated by Alizarin Red S, Oil red O, and Alcian blue staining, respectively.

The animals were anesthetized and transcardially perfused with 4% PFA. 8-mm segments of spinal cord at the center of injury site (or the same position in sham tissues) were collected and sectioned (30 µm thick). Immunohistochemistry of 4-Hydroxynonenal (4-HNE) and CTSB was conducted using the Super Plus™ High Sensitive and Rapid Immunohistochemical Kit (pH6.0) (Elabscience Biotechnology, E-IR-R221). The anti-4 HNE antibody and anti-CTSB antibody were used for labelling (Supplementary Table 2). Secondary antibody-only controls were employed to validate antibody specificity and distinguish genuine target staining from the background. Histopathological tissue staining was conducted using the Prussian Blue Iron staining kit (Solarbio Science & Technology, G1422) and Modified Oil Red O staining kit (Solarbio Science & Technology, G1261) following the manufacturer's instructions. The positively stained areas in the injured epicenter were quantitatively analyzed using the ImageJ software "IHC profiler."

MSCs grown to 80% confluency were incubated with Fe₃O₄ NPs (50 µg/mL) for 16 h, washed three times with PBS, and stained with a Prussian blue iron staining kit (Solarbio, Beijing, China), according to the manufacturer’s instructions.