*2.14. Confocal Microscope*

For mitochondrial morphology examination, the hepatocytes were incubated with 10 nM Mitotracker Red (Invitrogen Life Technologies, Carlsbad, CA, USA) for 45 min at 37 ◦C. After the indicated chemicals treatment, the hepatocytes were incubated with DAPI for another 45 min at 37 ◦C in the dark. The fluorescence images of each group were captured using Leica TCS SP5 II confocal spectral microscope. More than 20 clearly identifiable mitochondria were randomly selected from each treatment group. The length and density of the mitochondria were analyzed using Image J software.

To analyze Drp1 mitochondrial translocation, Cr(VI)-exposed hepatocytes were fixed with 4% paraformaldehyde for 15 min at room temperature following incubation with MitoTracker Red (10 nM, 45 min, 37 ◦C). The hepatocytes were then permeabilized with 0.5% Triton X-100, blocked with 5% bovine serum albumin (BSA), and incubated with primary Drp1 antibody at a 1:100 dilution at 4 ◦C overnight, followed by treatment with the secondary antibody. Nuclei were stained with DAPI prior to mounting. Confocal fluorescence images of each treatment group were captured with the Leica TCS SP5 II confocal spectral microscope.
