Fv3000 osr confocal system

Hela cells respectively transfected with WT ALDH18A1 plasmid and mutant ALDH18A1 plasmids were cultured in glass-bottomed dishes and mitochondria was visualized with MitoTracker red probe (Invitrogen). Then culture medium was removed from the transfected Hela cells and the cells were washed three times with ice-cold phosphate-buffered salin (PBS). The cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100, blocked with 5% donkey serum for 1 h, and immunostained with anti-His (1:1000) (Abmart) and secondary anti-mouse IgG Alexa Fluor488 antibody (1:500) (Life Technologies). Hela cells respectively transfected with WT AP5Z1 plasmid and mutant AP5Z1 plasmids were immunostained with anti-LAMP1 (1:1000) (Abcam) and secondary anti-mouse IgG Alexa Fluor488 antibody (1:500) (Life Technologies). Fluorescence images were captured by Olympus FV3000 OSR confocal system. More than 100 cells per visual field were quantified for each condition using Image-J software (NIH). Quantification of the particle number, fluorescence intensity and spot area was performed by a person blind to the experiment. Experiments were replicated three times.

Frozen muscle sections were fixed at room temperature in acetone for 10 min and washed in PBS. Nonspecific sites were blocked in PBS with 1% BSA and 0.01% Triton X‐100 for 1 h and incubated overnight at 4°C with rabbit anti‐ACTN2 (A8939; Abclonal). After washing, the sections were incubated 1h with the secondary antibody: Alexa Fluor 594 goat anti‐rabbit antibodies at room temperature. Cell nuclei were then stained with 40, 6‐diamidino‐2‐phenylindole (DAPI; Life Technologies). For C2C12 cell lines, after 24 h transfection with eGFP‐tagged ACTN2, cells were fixed at room temperature in 4% PFA for 10 min. DAPI was used for the staining of cell nuclei. Fluorescence images were captured by Olympus FV3000 OSR confocal system.