4 6 diamidino 2 phenylindole dapi

After being washed four times for 5 minutes in PBS, the sagittal frozen sections of MG were stained with neutral lipid droplet with HCS LipidTox solution (1:500, H34475; Invitrogen, Grand Island, NY, USA) for 30 minutes at room temperature. To visualize the nuclei of the cell, sections were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 1 µg/mL; Biofroxx, Einhausen, Germany) for 15 minutes. LipidTox staining images of representative areas of the MG were obtained with the confocal laser scanning microscope (LSM 980; Zeiss, Oberkochen, Germany).

The GC-2 cells were seeded on a cover slip and fixed with cooled 4% (w/v) formaldehyde at 4°C for 20 min. After blocking with 5% (v/v) normal goat serum (Beyotime) at room temperature for 1 h, the cells were incubated with the rabbit anti-PK2 antibody (1:200, Abcam) or rabbit anti-PKR1 antibody (1:500, Alomone) at 4°C overnight. After the cells were washed twice with PBS, the secondary antibody, goat anti-rabbit IgG H&L (FITC; 1:500, Abcam), was incubated with the cells at room temperature for 1 h. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; biofroxx, Einhausen, Germany). The fluorescence intensity was detected with a fluorescence microscope (Olympus).

HMGECs were seeded in 24-well plates at a density of 2 × 10⁴ cells/well until 80% confluency, and differentiation was then induced by MLT and LPS. For lipid analysis, cell slides were first fixed with 4% paraformaldehyde for 25 minutes at 4°C and then exposed to the neutral lipid droplet with HCS LipidTox solution (1:500; Invitrogen-Gibco) for 30 minutes. For lysosome staining, cells were further stained in a medium containing LysoTracker Deep Red (1:20,000; Invitrogen-Gibco) for 30 minutes. Slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 1 µg/mL; Biofroxx, Einhausen, Germany) for 15 minutes to allow nuclei visualization. Cell slides were observed and captured under the confocal laser scanning microscope (LSM 980; Zeiss, Oberkochen, Germany), and fluorescence intensities were quantified with ImageJ (National Institutes of Health, Bethesda, MD, USA).