Cells were fixed with 4% formaldehyde at room temperature for 45 min, followed by permeabilization for 3 min in ice-cold buffer (0.1% sodium citrate and 0.1% Triton X-100 in distilled water). Cells were blocked at room temperature for 1 h in DPBS with 1% BSA, and then incubated with Cpt1a mouse mAb (Abcam# ab128568), Bcl-2 rabbit pAb (ABclonal# A2845), Noxa rabbit pAb (ABclonal# A9801), or Puma rabbit pAb (ABclonal# A17138). All images o were quantitated using Image J. All data were calculated as the intensity units arbitrary to corresponding DAPI intensity or as fold change to control.
For confocal analysis of colocalization between MitoTracker Red and Bcl-2, cells were first stained with MitoTracker Red (250 nM, 20 min), and then fixed with stained with Bcl-2 antibody, as described above.
For confocal analysis of colocalization between MitoTracker Red and Bcl-2, cells were first stained with MitoTracker Red (250 nM, 20 min), and then fixed with stained with Bcl-2 antibody, as described above.