4 6 diamidino 2 phenylindole (dapi)

Approximately 1 × 10⁶ cells/well were seeded in 24-well plates. After being attached, PGCs were treated with 10 nM 1α,25(OH)₂D₃, chloroquine (10 μM), and NAC (4 mM) for 24 h. MDC (monodansylcadaverine) was used as a tracer of autophagic vesicles. The autophagosomes are marked as clear green dots under the fluorescence microscope. After treatment, the cells were treated with MDC (0.05 mM) (Kaiji Biotechnology Co., Ltd., Nanjing, China) and DAPI (1 μg/mL) (4′,6-diamidino-2-phenylindole) (Solarbio) following the manufacturer’s protocol. Briefly, the cells were grown with MDC and DAPI at 37 °C for 15 min and fixed immediately with paraformaldehyde (4%) in PBS for 20 min, then observed under a fluorescence microscope (Olympus Corporation). LED intensity, integration time, and camera gain were fixed while taking pictures. Image J software was used to process the images, a total of 200 cells in each sample were analyzed, and the percentage of cells with green spots indicates the percentage of autophagy.

HUVECs were cultivated to confluence on glass slipcovers before their fixation with 4% paraformaldehyde for 10 min. Fluorescent labeling antibodies were applied to monolayer cells after blocking with 3% BSA for 30 min at room temperature. Afterwards, sections were incubated with DAPI (Solarbio, China), a nucleus-specific anti-fluorescence agent.
Tissues embedded in paraffin were sectioned and placed on glass transparencies. Sections were immersed in 3% H₂O₂ and serum albumin to inhibit endogenous peroxidase activity and nonspecific binding, respectively. After that, primary antibodies and secondary antibodies conjugated with Alexa Fluor 488 or 594 (Abcam, USA) were added, and the sections were incubated at 4 °C for 24 h. Anti-fluorescence quenching agent DAPI (Solarbio, China) was used to counterstitch nuclei. Image acquisition was performed using a Zeiss LSM510 Meta (Zeiss, Germany).

mEXOs were incubated with a red fluorescent dye (Dil, Biotium, USA) for 30 min, and then were centrifuged to remove contaminating dye and to obtain the labeled mEXOs. For internalization assay, HUVECs were seeded in 24-well culture plates (30,000 cells per well) for 12 h, and co-cultured with Dil-labeled mEXOs for 24 h. After incubation, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 10 min; thereafter, the nucleic was stained with DAPI (Solarbio, Beijing, China) and the cytoskeleton was stained with FITC phalloidin (Yeasen Biotech Co., Shanghai, China) according to the manufacturer’s instructions. The mEXO uptake by cells was observed by using the laser scanning confocal microscope. For cell miR-31-5p uptake assay, FAM-labeled miR-31-5p mimics were obtained from Ribobio company (Guangzhou, China) and were encapsulated into Dil-labeled mEXOs through electroporation. HUVECs were seeded in 24-well culture plates (30,000 cells per well) for 12 h, and co-cultured with PBS, FAM-labeled miR-31-5p mimics, Dil-labeled mEXOs, and FAM-Dil-labeled mEXO-31, respectively for 24 h. After incubation, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 10 min; thereafter, the nucleic was stained with DAPI (Solarbio, Beijing, China). The cell miR-31-5p uptake was observed by using the laser scanning confocal microscope.