Sp8 model confocal microscope

Cells were stained with MitoTracker Red (Thermo Fisher Scientific) for 30 minutes at 37°C, washed with PBS, and fixed with 2% paraformaldehyde in PBS for 15 minutes at room temperature. After washing in 10mM glycine in PBS and blocking with 5% normal goat serum (Invitrogen) in PBS, the cells were permeabilized with 0.5% saponin (Sigma) in 5% normal goat serum in PBS for 1 hour at room temperature. Staining was performed in permeabilization buffer. Primary antibodies used were the same as used for western blotting, except that a rabbit antiserum against calnexin was used. Secondary antibodies were the Alexa Fluor series from Thermo Fisher Scientific. Slides were mounted with ProLong Gold Anti-Fade Reagent (Thermo Fisher Scientific), viewed using a Leica SP8 model confocal microscope and the images were analysed using ImageJ software.

Cells were grown on glass cover slips and following indicated treatment were washed with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature followed by three washes with PBS. Cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature followed by three washes with PBS and blocked with 5% FBS, 0.05 M glycine in PBST (0.5% Tween-20 in PBS). Primary and secondary staining were performed in 5% FBS, 0.05 M glycine in PBST. Primary antibodies used were the same as used for immunoblotting. Secondary antibodies were the Alexa Fluor series from Thermo Fisher Scientific. Nuclear chromatin staining was performed by incubation in blocking solution containing 0.5 mg/mL 4’,6-diamidino-2-phenylindole, DAPI (Sigma-Aldrich). Slides were mounted with ProLong Gold Anti-Fade Reagent (Thermo Fisher Scientific), imaged on a Leica SP8 model confocal microscope and analyzed using ImageJ. For mitochondria staining, cells were pre-stained with MitoTracker Red (Thermo Fisher Scientific) for 45 min at 37°C before starting the procedure.