4 6 diamidino 2 phenylindole (dapi)

Frozen tissue was sectioned with a frozen microtome (Leica) and stained with DAPI (diamidino-phenyl-indole) under dark conditions. An antifluorescence quencher was added dropwise; the tissue sections were sealed with glass coverslips; and the cells were observed under a fluorescence microscope (Olympus Corporation). The presence of green fluorescence in frozen sections after excitation with blue light indicated positive transfection. The transfection rate was the proportion of green fluorescence cells to the general count of cells (green fluorescence protein/DAPI × 100%).³⁹ (link)^,40 (link)

Cells on the coverslips were fixed with 4% paraformaldehyde and incubated with the primary antibody against PARK2 (Santa cruz, sc-32282), YAP (CST, 14074) at 4 °C overnight. After washing with PBS, cells were then incubated with fluorescence-conjugated secondary antibody (Invitrogen, Carlsbad, CA), and subsequently counterstained with DAPI (Life Technology). Images were captured after staining with anti-fade DAPI solution using a confocal laser-scanning microscope (Leica TCS SP8 STED). The fluorescence-integrated density was measured by ImageJ software and the mander's co-localization coefficients were generated by Zen software.

The membranes were cropped into round samples 1 cm in diameter, and samples from the different groups were plated on 48-well plates (Jet Biofil, China). The cells were then seeded at 5 × 10³ cells per membrane. After culturing for 3 days, FITC phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) staining was performed to stain the nuclei and F-actin of the cells, which were observed under a confocal laser scanning microscope (CLSM, Leica TCS SP8 X, Germany). After removing the culture media, the samples were washed with phosphate-buffered solution (PBS) before fixing the cells with a 4% paraformaldehyde (PFA) solution. Triton X-100 solution (0.5%) was used to permeabilize cells for 10 min, and the samples were washed again with PBS. FITC phalloidin (Yeasen, China) was diluted with 1% bovine serum albumin (BSA) solution at a ratio of 1:200. The solution was then added to the cells and incubated at room temperature for 1 h. After three rounds of washing for 5 min, 100× diluted DAPI (Yeasen, China) solution was added to the samples for nuclear staining and incubated for 10 min. After cleaning the residual dye with PBS, the membranes were transferred to confocal dishes for observation.

Cell cytoskeleton experiment was performed with 4′,6-diamidino-2-phenylindole (DAPI, Boster, Wuhan, China) and phalloidin (TraKine^TM F-actin Staining Kit, Abbkine, Wuhan, China) after 7 days of chondrogenic culture. The BMSCs-loaded constructs were fixed with ice-cold 4% formaldehyde for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, incubated for 20 min in 1% bovine serum albumin in PBS, for 45 min in green fluorescence dye-labeled phalloidin to stain the f-actin and for 15 min in DAPI to stain the nuclei all at room temperature, and photographed using a confocal laser scanning microscope (CLSM, Leica TCS SP8, Germany).

Terminal‐deoxynucleotidyl Transferase Mediated Nick End Labeling (TUNEL) staining in cultured spinal cord neurons were performed. Briefly, the samples were collected on the 3rd days after transfection, cultured neurons were fixed with 4% paraformaldehyde (Cat# 142287, Beyotime, Shanghai, China) for 20 min at room temperature, then rinsed with PBS (Cat# BL551A, Biosharp, Hefei, China) for 20 min. Subsequently, cells incubated with TUNEL reaction mixture (Cat# Rs‐11684817910, In situ Cell Death Detection Kit, Roche Molecular Biochemicals, Mannheim, Germany) for 1 h at 37°C, then rinsed with PBS for 3 times and finally incubated with DAPI (Cat# C1005, Beyotime Biotechnology, Shanghai, China) for 5 min. Five fields were acquired, and apoptosis was quantified by determining the percentage of TUNEL/DAPI with Leica AF6000 DMI6000B (LAS AF system).

Apoptosis in the heart tissue was detected by the terminal deoxynucleotidyl transferase-mediated FITC-dUDP nick-end labeling (TUNEL) method. Heart tissue sections were dewaxed and incubated with 3% H₂O₂ for 20 min at room temperature. The reaction mixture (TUN11684817, Roche, Basel, Switzerland) was dropped onto slides and incubated at 37°C for 60 min. After the sections were rinsed 3 times, they were incubated in DAPI (2 mg/ml, Solarbio, Beijing, China) for 5 min. Finally, the number of TUNEL-positive/DAPI-stained apoptotic bodies was counted with an electric light microscope (DM3000, Leica, Solms, Germany).