Alexa fluor rabbit 568

Three days after the 20 μM PMO transfection, 10,000 patient fibroblasts seeded onto glass cover slips were treated with 50 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 2 h to depolarise the mitochondria. The glass cover slips were collected and fixed in acetone/methanol (1:1) on ice and 20% goat serum in PBS-T (0.2% Triton-X in 1× PBS) was used to block the slides for 1 h. Parkin was probed with MAB5512 (1: 200, Merck) for 1 h and Alexa Fluor mouse 488 (1:400, Thermo Fisher Scientific) for a further hour. Tomm20 was probed with HPA011562 (1:100, Sigma-Aldrich) at room temperature for 1 h and Alexa Fluor rabbit 568 (1:400, Thermo Fisher Scientific) for another hour. Nuclei were stained with Hoechst (1:160, Sigma-Aldrich). Images were captured and analysed using the Nikon Eclipse 80i microscope with the NIS elements program.

TIG3 cells were treated with 500 mM sorbitol for 3 h and fixed in 4% formaldehyde for 10 min. Fixed cells were incubated for 1 h at RT in IF buffer (3% BSA, 0.1% Triton-X) with primary antibodies: keima anti-mouse (MBL M126–3M, 1:200) and fibrillarin anti-rabbit (abcam ab5821, 1:200). Cells were washed 3x in PBS and incubated for 1 h at RT in IF buffer with secondary antibodies: Alexa Fluor rabbit-568 (Thermo Fisher A10042, 1:1000) and Alexa Fluor mouse-488 (Thermo Fisher A11029, 1:1000). Cells were stained with DAPI (Sigma-Aldrich 10236276001) for 10 min and mounted in DAKO mounting medium (S3023). Cells were imaged with a Zeiss LSM800 confocal microscope with 63x/1.4 oil DIC objective using Zeiss Zen software.