Gdp1024

The aortic root sections underwent dehydration and blocking with 3% BSA prior to immunofluorescence staining. Subsequently, the sections were exposed to anti-CD68 antibody (1:200 dilution, GB113109, Servicebio) overnight at 4 °C, followed by incubation with Cy3-conjugated Goat Anti-Rabbit IgG (H+L) secondary antibody (1:300 dilution, GB21303, Servicebio) at room temperature for one hour in the absence of light. DAPI (1:1000, GDP1024, Servicebio) was utilized for nuclear counterstaining, with 10 min of incubation at room temperature in the dark. Images were scanned using an automatic digital slide scanner (3DHISTECH Pannoramic MIDI, Budapest, Hungary). Aipathwell software (version 1.0, Servicebio, Wuhan, China) was used to calculate the rate of positive cells.

For antigen retrieval, paraffin-embedded gastrocnemius muscle sections were heated at 121 °C for 10 min. Sections were blocked with 5% donkey serum (G1217, Servicebio, Wuhan, China) and incubated with primary antibodies at 4 °C overnight. Finally, sections were incubated with fluorescent secondary antibodies for 1 h and DAPI (GDP1024, Servicebio, Wuhan, China) for 15 min. Immunofluorescence was visualized and imaged using a fluorescence microscope (Olympus, Tokyo, Japan). Antibodies used in the immunofluorescence assay were against P4HA1 (1:100, 12658-1-AP, Proteintech), NG2 (1:200, ab275024, Abcam, Cambridge, UK), Myosin (1:200, ab37484; Abcam, Cambridge, UK), CD31 (1:400, ab182981; Abcam, Cambridge, UK), and Flag (1:200, MA1-91878, Thermo Scientific, USA).