Prussian blue staining kit

The deposition of iron-containing heme in lung tissue was detected by using Prussian blue staining kit (Solarbio, China). Paraffin sections with a thickness of 4 µm were prepared, routinely dehydrated and transparent, and then stained with Perls staining solution for 30 min. The sections were washed three times with distilled water; the background was lightly stained with Eosin staining solution for 15–30 s, and rinsed with tap water for 2–3 s. The sections were dehydrated with anhydrous ethanol and transparent with xylene, sealed with neutral gum, and placed under a light microscope for observation. The mean percentage positive area was used to quantify the difference between the two groups of Prussian blue results.23 (link) This is a commonly used method of immunohistochemical analysis that counts the percentage of positive staining area over the measured area and finally takes the mean value as the percentage value of the mean positive staining area for this case section, thus being less affected by some human-induced interference. Color threshold quantification was performed on six non-overlapping areas/groups by image J 1.53 software (NIH, USA). All images were evaluated by researchers who were unaware of the experimental group.

MSCs were cultured to achieve an 80% fusion rate. Different Fe₃O₄@PDA NP concentrations were added to MSC culture plates followed by incubation for 24 h. Next, the cells were washed thrice with PBS and fixed using 4% paraformaldehyde for 10 min. Subsequently, MSCs were treated based on the instructions of the Prussian blue staining kit (Beijing Solarbio Science & Technology Co., Ltd.). After staining, the NP density in the cells was observed using an inverted optical microscope (Olympus, Tokyo, Japan).
MSCs were co-cultured with Fe₃O₄@PDA NPs at 50ug/mL for 24 h. Next, the cells were digested with trypsin, washed with PBS, and re-suspended thrice. This was followed by centrifuging the cells for 10 minutes at 1000r, discarding the supernatant, adding electron microscope fixative solution, and fixing at 4°C for 24 hours. We determined the ultrastructural characteristics of Fe₃O₄@PDA NPs in cells using transmission electron microscopy (FEI Czech Republic s.r.o, Netherlands).

After the ABR measurement, the cochlea from adult mice were fixed in 4% paraformaldehyde solution overnight and then decalcified in 0.5 M ethylene-diamine-tetraacetic acid (EDTA) for 2 days. All outer bones were removed from the decalcified cochlea under the microscope, and the stria vascularis (SV) and modiolus were separated from the organ of Corti (OC) where the hair cells were located. The tissues were stained using a Prussian blue staining kit (Solarbio, Beijing, China).

Curcumin (Cur) and dopamine hydrochloride (DA) all purchased from Aladdin. Maleimide-PEG2k-Fmoc (Fmoc-PEG-MAL) was purchased from Xi'an Ruixi Biological Technology (Xi'an, China). DMEM high glucose medium, trypsin, streptomycin, and glutamine were obtained from Hyclone. Fetal bovine serum (FBS) was supplied by Tianhang Biotech. ABTS reagent 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was sourced from Shanghai McLean Biochemical Technology Co., Ltd. Propidium iodide/acridine orange staining kit was provided by Biyuntian in China, Shanghai. MTT assay kit, MDA content detection kit, SOD activity assay kit, Prussian blue staining kit, and Fe²⁺ ion content detection kit were all procured from Solarbio Life Sciences. ROS detection assay kit, BCA protein concentration determination assay kit, and LPO assay kit were acquired from Biyuntian Biotech.