C1002

Cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Then cells were permeabilized with 0.5% Triton X‐100 for 20 min at room temperature, blocked and incubated with anti‐NSUN2 (1:200, Proteintech, 20854‐1‐AP), anti‐m⁵C (1:100, abcam, ab10805), or anti‐Ki67 (1:100, CST, 9449) at 4°C overnight. Afterwards, cells were washed and incubated with fluorochrome‐conjugated secondary antibody (1:500) for 1 h at room temperature followed by DAPI (Beyotime, C1002) staining. Images were acquired with confocal microscopy.

The concentration of PBMCs suspension was adjusted to 5 × 10⁶/mL with PBS, and 20 µL of PBMCs suspension for each sample was dripped onto glass slides, fixed with 4% paraformaldehyde, and sealed with 0.1% TritonX-100 and 1% bovine serum albumin (BSA). Rabbit polyclonal antibody to TRIM7 (LS-C146201, LifeSpan Biosciences) was used for the primary antibody, Goat anti-Rabbit IgG-FITC antibody (HA1004, Hangzhou HuaAn Biotechnology) was used for the secondary antibody, and 4′,6-diamidino-2-phenylindole (DAPI) staining solution (C1002, Beyotime) was used for staining cell nucleus. After sealing by Prolong, the slides were observed and photographed under a laser confocal fluorescence microscope (LSM710, Zeiss). We used ImageJ software to analyze the brightness of the cells in the immunofluorescence maps, and randomly selected 20 cells in each group for quantitative detection and took their mean values for comparison.

The paraffin sections were deparaffinized to water and then subjected to proteinase K (ST533, Beyotime, China) repair, followed by membrane rupture with a membrane-breaking fluid (G1204, Servicebio, Wuhan, China) and the addition of a TUNEL reaction solution (C1088, Beyotime, China). Finally, the sections were sealed after DAPI (C1002, Beyotime, China) counterstaining. Pictures were taken with a positive fluorescence microscope (NIKON ECLIPSE C1, Japan), and they were quantified using Image J.

To examine cell morphology, the cells of dTHP-1 were incubated with 500 nM CytK and then observed with a microscope (Ti-S/L100, Nikon). The movie was processed using Image-Pro Plus 6.0. To examine cell membrane integrity, the cell culture was supplemented with propidium iodide (2 μg/ml) (PI, ST511, Beyotime), and observations were conducted with a confocal microscope (Zeiss LSM 710). The percent of PI-positive cells was calculated at least from five random fields for each experiment. During immunofluorescence microscopy, the cells of dTHP-1 were treated with CytK (500 nM) for 25 min, washed in PBS three times, and then treated with 4% paraformaldehyde for 10 min. After washing as above, the cells were incubated with permeabilization solution (0.2% Triton X-100 in PBS) for 5 min and blocked with 5% BSA for 1 h. The cells were incubated with rabbit anti-CytK (1:100 dilution in 1% BSA) overnight at 4 °C and treated with goat-anti-rabbit antibody (Alexa Fluor 488) (ab150081, Abcam) (1:500 dilution) for 1 h, followed by DAPI (C1002, Beyotime) staining for 10 min. After each incubation step, 0.1% Tween-20 (in PBS) was used to wash the cells. A confocal microscope (Zeiss LSM 710) was used to observe the cells. Scanning electron microscopy was performed as reported previously [47 (link)].

The treated cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 (ST795, Beyotime Biotechnology, Jiangsu, China) for 5 min. The slices were blocked in 5% BSA at 25°C for 1 h, and subsequently incubated with primary antibodies against podocin (ab50339, Abcam, Cambridge, UK, 1:200) and nephrin (ABIN2177857, antibodies-online.com, Aachen, Germany, 1:200), and synaptopodin (ab224491, Abcam, 1:100) at 4°C overnight. The following day, sections were stained with horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-rabbit IgG (BA1032, Boster Biological Technology, Wuhan, China), and the cell nucleus was stained by DAPI (C1002, Beyotime Biotechnology).

The brain slices were washed 3 times with PBS for 5 min each time and then treated with 0.1% Triton X-100 (Beyotime Biotechnology, ST795, Shanghai, China) diluted with PBS for 20 min. Then, slices were placed in 5% BSA (Beyotime Biotechnology, ST023, Shanghai, China) diluted with PBS and sealed at room temperature for 1 h. After incubating overnight with the primary antibody [rabbit antibody PSD95 (Abcam, ab238135, Cambridge, UK, 1:200), rabbit antibody IBA1 (Fujifilm, 019-19741, Tokyo, Japan, 1:300)] at 4 °C, the sections were washed 3 times with PBS. A secondary antibody [goat anti-rabbit IgG H&L (Alexa Fluor^® 488) (Abcam, ab150077, 1:500)] was then applied at room temperature for 1 h, and the slices were washed in PBS 3 times. Finally, the nuclei were restained with 1 μg/mL DAPI (Beyotime Biotechnology, C1002, Shanghai, China) for 10 min, and the slides were mounted with an anti-fluorescence quenching seal. The slides were viewed with a fluorescence microscope (BioTek, Cytation5, Hong Kong, China) and all images were collected using a microscopic imaging system (BioTek, Cytation5, Hong Kong, China). The ImageJ (v1.54f) application was used to analyze the collected images.

Mice were anesthetized with isoflurane and then intracardially perfused with PBS and 4% PFA (Paraformaldehyde, Merck, Darmstadt, Germany) in PBS. The brain was removed and post-fixed in PFA for two days. The coronal brain sections (40 μm) were collected using a Thermo Fisher cryostat microtome (NX50, U.S.A) for the M1 and the whole brain with H8 injection. The sagittal cerebellum sections were collected for the 5Cb. To stain for CaMKIIα and D-28K, brain sections were washed three times with PBS and incubated with blocking solution for two hrs at room temperature. The sections were incubated with anti-CaMKIIα goat antibodies (1:500, ab87597, Abcam) or anti-D-28K mouse antibodies (1:500, C9848, Merck) at 4 °C for 48 hrs. Then the slices were washed with PBS for three times, and incubated with Cy3 rabbit anti-goat (1:500, 106831, Jackson) or Cy3 rabbit anti-mouse (1:500, 315-165-049, Jackson) for one hour at 37 °C. The slices for visualization of NE1h, GABA and Glu sensors were directly washed with PBS for three times. All slices were counterstained with DAPI (1:4000, C1002, Beyotime) before imaging on a microscope (Olympus VS120, Japan) using 10× and 40× objectives.