Secondary anti mouse igg alexa fluor488 antibody

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.