G1217

Different treatments for the U251 cells were applied according to the manufacturer’s protocols after 24 h. First, the intracellular Ca²⁺ with Fluo-4 AM was fluorescently stained and then incubated with MitoSOX (5 μM) at 37 °C for 10 min in the dark, washed twice with Hanks with calcium magnesium (1 × HBSS, PH1509, Phygene) pH 7.4, and pre-cooled at 4 °C in 4% paraformaldehyde and fixed for 10 min at RT. Other U251 cells were stained with Fluo-4 AM and then blocked with 10% normal donkey serum (G1217, Servicebio, Wuhan, China) for 30 min at RT and were then incubated with rabbit anti-Piezo1 antibody (1:200, 15939-1-AP, Proteintech, WuHan, China) at 4 °C overnight (about 16 h). The next day, the fluorescence-labeled secondary antibodies were added (1:400, 711-545-152, Alexa Fluor 488-ChromPure Donkey IgG, Jackson ImmunoResearch, West Grove, PA, USA; according to the instructions, 0.5 mg was rehydrated with 0.4 mL ddH₂O at an antibody concentration 1.5 mg/mL) for 1 h at RT in the dark. Finally, they were washed with PBS three times (5 min each time) and were then incubated with DAPI (G1012, Servicebio) at RT in the dark for 15 min and then washed three times again. The cell nucleus was stained with Hoechst 33342 (CSA: C1028, Beyotime) for 10 min at 37 °C in the dark, and images were captured with a confocal microscope (ZEISS LSM 880, Oberkochen, Germany) (×40 oil immersion lens).

At the end of the animal experiments, all experimental C57BL/6 mice were sacrificed, and the brains were completely removed and were perfused with 4% paraformaldehyde and were immersed for one week and then dehydrated with gradient sucrose for 48 h, embedded in optimal cutting temperature compound (OCT) (SAKURA, CA, USA), frozen and sectioned into portions that were 35 μm thick, and stored in antifreeze buffer for testing. The brain slices were taken from the antifreeze buffer and washed twice with PBS and then blocked with 10% normal donkey serum (G1217, Servicebio) for 1 h at RT followed by incubation in rabbit anti-CD86 antibody (1:200, DF6332, Affinity) and rabbit anti-MRC1 (CD206) antibody (1:200, DF4149, Affinity) at 4 °C overnight (about 16 h). The next day, the fluorescence-labeled secondary antibodies (1:400, 711-545-152, Alexa Fluor^® 488-ChromPure Donkey IgG, Jackson ImmunoResearch) were added for 2 h at RT in the dark. Finally, they were washed with PBS three times (5 min/time) and were incubated with DAPI (G1012, Servicebio) at RT in the dark for 15 min and then washed three times again. Images were captured with a confocal microscope (ZEISS LSM 880, Oberkochen, Germany) (×40 oil immersion lens).

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).