Ix73p2f

The organic element content was determined using elemental analysis (EA) (EA 2400 II, PerkinElmer, USA). Because the nitrogen content of various proteins was 16%, the amount of protein in the samples was obtained by multiplying the percentage of nitrogen element by 6.25 [36 (link)]. The morphology, structure, and elemental maps of the samples were determined by scanning electron microscopy (SEM) (Sigma 300, Zeiss, German) with energy dispersive X-ray spectroscopy (EDS). A fluorescence microscope (IX73P2F, Olympus, Japan) was used to collect optical and fluorescence microscopy images. Other characteristics were determined using Fourier transform infrared spectroscopy (FT-IR) (Vertex 70, Bruker, USA), X-ray photoelectron spectroscopy (XPS) (PHI-5000 Versaprobe III, ULVAC-PHI, Japan), X-ray diffraction (XRD) (SmartLab, Rigaku, Japan), and flow cytometry (FCM) (MoFlo Astrios EQ, Beckman Coulter, USA).

Gastrocnemius muscles were fixed in 10% buffered formalin for 24 h at 4 °C, dehydrated through graded alcohols, and embedded in paraffin. The muscles were sectioned into 5 μm slices using a microtome (Leica RM2235, Wetzlar, Germany). Hematoxylin-eosin (H & E) and Sirius red (G1470, Solarbio, Beijing, China) staining were performed to examine histological alteration and collagen deposition, respectively. The procedure was performed according to the manufacturer’s protocol. Before staining, paraffin sections were deparaffinized using xylene and hydrated with distilled water. For H & E staining, section-mounted glass slides were stained in hematoxylin for 5 min and then washed in distilled water. Slides were further immersed in an eosin solution for 1 min and washed in distilled water for 10 min. After 1 min, the slides were sequentially immersed in a series of ethanol solutions (70%, 95%, and 100%), for dehydration. After immersion in xylene for 3 min twice, the slides were fixed with neutral resin and sealed with cover slip. For Sirius red staining, we used the Mayer hematoxylin for 10 min, followed by washing. Then the collagen was stained by Sirius red for 1 h. The other steps were the same as the H & E staining’s processes. Images were captured with an Olympus microscope system (IX73P2F, Olympus, Tokyo, Japan).

A RAW264.7 macrophage suspension with a cell density of 1 × 10⁵ cells/mL was plated on a 6–well plate with round coverslips for 12 h to allow it to adhere to the plate. A prepared OVA–FITC was mixed with PHP, PHP1, and PHP2. The mixtures were added to the culture plate and incubated for 12 h. The coverslips were taken out and fixed with 4% paraformaldehyde for 30 min, stained with the DAPI reagent for 20 min, followed by staining with DID for 20 min. The samples were washed thoroughly with PBS after each staining. Finally, the coverslips were covered with 90% glycerol and observed under a fluorescence microscope (IX73P2F, Olympus, Tokyo, Japan). The rest of the cells were scraped off and examined via flow cytometry (CyFlow Cube8, Sysmex, Norderstedt, Germany).