Diamidine phenylindole dapi

We detected a specific autophagy marker for LC3-positive MSCs using immunofluorescence staining. MSCs were washed twice with PBS. Then, they were fixed with 4% paraformaldehyde in PBS for 30 min, and they were permeabilized with methanol. The MSCs were treated with 3% hydrogen peroxide in methanol for 15 min and washed with tap water for 15 min. The cells were incubated with 0.5% Triton X-100 (KeyGEN) for 5 min, and then, they were washed and incubated with blocking solution (10% goat serum in PBS) for 30 min. Next, they were treated with anti-LC3B antibody (1:200 dilution; Novus Biological, Littleton, CO, USA) overnight at 4 °C. After washing with blocking solution 3 times, the cells were incubated with secondary antibodies. The cells were stained with diamidine phenylindole (DAPI) (Molecular Probes, Waltham, MA, USA), and they were imaged using a confocal microscope (Dmi 6000-B, Leica, Brunswick, Germany).

Transmission electron microscopy (TEM) (JEM-1011, JEOL, Akishima, Tokyo, Japan) was used to observe the autophagosomes of MSCs, as described previously.^{14 (link)}
The morphological characteristics of autophagosomes include a crescent or cup-shaped bilayer or multilayer membrane, with a tendency to enclose cytoplasmic components. Immunofluorescence analysis was performed to detect a specific autophagy marker LC3, as described previously.^{14 (link)}
Briefly, the cells were washed with phosphate-buffered saline (PBS) (KeyGEN) and fixed with 4% paraformaldehyde. Then, the cells were again washed with PBS and incubated with 0.5% Triton X-100 (KeyGEN). Subsequently, the cells were incubated with a blocking solution (10% goat serum in PBS) and then treated with the anti-LC3B antibody (Novus Biological, Littleton, CO, USA) overnight at 4 °C. Finally, the cells were incubated with secondary antibodies after washing with the blocking solution. The MSCs were stained with diamidine phenylindole (DAPI) (Molecular Probes, Waltham, MA, USA) and observed under a confocal microscope (Dmi 6000-B, Leica, Brunswick, Germany). Western blot analysis was used to detect the autophagy makers Beclin1 and LC3, as described previously.^{14 (link)}