Mouse anti cytochrome c

Cells were harvested, followed by fixation with 4% PFA and permeabilization by 0.5% Triton X. in situ Proximity ligation assay (PLA) was performed by using Duolink PLA reagents (MilliporeSigma, Burlington, MA, USA) according to the manufacturer’s instructions. Cells were blocked with Duolink in situ blocking solution for 1 h before being probed with primary antibodies in Duolink in situ antibody diluent overnight at 4 °C. Cells were then incubated with oligonucleotides-conjugates secondary antibodies (PLA probe anti-rabbit PLUS and anti-mouse MINUS), followed by ligation and amplification with the fluorophore-labeled oligonucleotide probe (excitation = 495 nm, emission = 527 nm). For immunofluorescence staining, cells were incubated with primary antibodies overnight and then labeled with secondary antibodies for 1 h. Cells were stained with DAPI and the fluorescent images were developed by Keyence BZ-X700-All-in-one Fluorescence microscope (Keyence). Primary antibodies for PLA and immunofluorescent staining were as follows: mouse anti-VDAC1 (820701, BioLegend, San Diego, CA, USA), rabbit anti-IP3R1 (A302-158, Bethyl, Montgomery, TX, USA), mouse anti-Cytochrome c (612503, BioLegend), and rabbit anti-HSP60 (12165, CellSignaling).