Mouse anti cytochrome c antibody

For a double immunocytochemical labeling, cells were fixed with 4% paraformaldehyde (EMS, Hatfield, PA, USA) at RT for 10 min at various time periods after drug treatment. For the images of TRAP1 and cytochrome c, cells were permeabilized with 0.3% saponin (Sigma) at RT for 10 min followed by blocking in 0.2% cold water fish gelatin/0.5% BSA in PBS (PBG) at RT for 30 min. After permeabilization and blocking steps, cells were incubated with a mouse anti-TRAP1 antibody, mouse anti-cytochrome C antibody (BD Biosciences; 1:200) or rabbit anti-Tom20 (Santa Cruz; 1:200) in 0.2% PBG overnight at 4℃. After extensive washing, cells were incubated with Alexa488-conjugated goat anti-rabbit IgG (Invitrogen; 1:200) and Alexa568-conjugated goat anti-mouse IgG (Invitrogen; 1:200) at RT for 1 hr. Hoechst 33258 staining (Molecular Probes; 1 µg/ml, Eugene, OR, USA) was performed for 10 min at RT. Slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Samples were observed under an LSM 510 META confocal laser scanning microscope equipped with epifluorescence and a digital image analyzer (Carl Zeiss, Zena, Germany).

Cytochrome c release was assessed as previously described (44 (link)). Cells were incubated in 1:200 mouse anti–cytochrome c antibody (catalog no. 556432; BD Pharmingen) and 1:200 PE Rat anti-mouse IgG1 (immunoglobulin G1) secondary antibody (catalog no. 550083; BD Pharmingen).