Triton

OC tissues were immobilized using 4% paraformaldehyde and permeabilized via 0.5% Triton (Beyotime, Jiangsu, China). After washing three times with PBS, 5% BSA was used to block the tissues. OC tissues were then incubated with an anti-LC3 primary antibody (1:50; Cat No. 14600-1-AP, Proteintech, Wuhan, China) at 4 °C overnight. Then, a FITC conjunct-second antibody was incubated with OC tissues for 2 h, and DAPI was used to label cell nuclei. Finally, a 90i fluorescence microscope (Nikon, Japan) was used to obtain the fluorescence signal of the LC3 dots in each OC tissue.

The cells were fixed with 4% paraformaldehyde (Beyotime) for 30 min, treated with 0.5% Triton (Beyotime) for 10 min, then closed with 0.5% BSA (Solarbio) for 30 min at room temperature. The closure solution was discarded, and the cells were then incubated with the primary antibody overnight at 4 °C. The cells were treated for one hour at room temperature with fluorescent secondary antibody dilution (Abbkine), shielded from light, and washed three times with PBS. PBS was blotted dry, and appropriate amount of anti-fluorescence quencher (Solarbio) was added to cover the bottom of the confocal dish. Place it under a confocal microscope, observe and take pictures. Images were analyzed using Image J.

Four groups were chosen for the cytotoxicity assay: (1) TGF-β3-PLGANPs: supplemented with the TGF-β3-loaded PLGANPs at the concentration of 1000 μg/mL in the culture medium; (2) TGF-β3 group: supplemented with the TGF-β3 at the concentration of 10 ng/mL in the culture medium; (3) PLGANPs group: supplemented with the blank PLGANPs at the concentration of 1000 μg/mL in the culture medium; (4) control group: no supplement in the culture medium. Except for the TGF-β3 group, the MSCs or MSCs-seeded hybrids were cultured with the complete culture medium. Cells cultured with 0.1% Triton (Beyotime Biotechnology, China) were chosen as negative control. In order to evaluate the cytotoxicity of the PLGANPs, MSCs were seeded on the 24-well plates at the density of 1 × 10⁴/well. After 1 day, the culture medium was removed and 1 mL culture medium containing the PLGANPs at the concentration of 1000 μg/mL was added to the wells after the cell attachment. The cytotoxicity was evaluated after 7 days' coculture in vitro by a LIVE/DEAD Viability/Cytotoxicity Assay Kit (Invitrogen, USA) according to the manufacturer's instructions. The stained cells were observed with a fluorescent microscope (LSM 510, Zeiss, Germany). Living cells percentage was calculated by Image J software (Wayne Rasband, National Institute of Health, USA). Three images from three samples were evaluated for each group.