Anti pink1 ab23707

NP cells were plated on glass coverslips and treated with 100 nM MitoTracker™ Red CMXRos (Thermo Fisher, M7512) for 30 min after completion of the experimental treatments. Cells were then fixed with 4% PFA or ice cold methanol for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. and blocked with 1% BSA for 1 h. Cells were incubated with anti-BNIP3 (3769S), anti-BNIP3L (12396S), anti-LC3 (12741) (Cell Signaling), anti-TOMM20 (ab186734), anti-LAMP1 (ab24170), anti-PINK1 (ab23707) (Abcam), anti-PARKIN (sc-32282) (Santa Cruz), anti-DRP1 (611113), anti-OPA1 (612607) (BD Biosciences), anti-BECLIN1 (NB500–249) (Novus Biologicals) in blocking buffer at 1:100–200 at 4°C overnight. After washing, cells were incubated with Alexa Flour-488 or Alexa Flour 647 and mounted with ProLong Gold Antifade Mountant with DAPI. Negative controls were used to confirm the specificity of staining (Suppl. Fig. 4). Cells were visualized using a Nikon A1R confocal microscope using 60x objective (CFI plan Apo Lambda 60x/1.40 oil). Positive staining for markers and co-localization of LC3 and LAMP1 with mitochondria was measured as area (pixel²/cell) using the colocalization Plugin of ImageJ software (http://rsb.info.nih.gov/ij/).