Rabbit anti tom20 antibody

Approximately 200 oocytes were lysed in RIPA buffer (solarbio, Beijing, China) supplemented with 1 mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF, solarbio) on ice for 30 min. Samples were boiled at 100°C in a metal bath for 10 min in protein loading buffer (CoWin Biosciences, Beijing, China) and equal amount of proteins were separated by 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, USA). After transfer, the membranes were blocked in TBST that contained 3% BSA for 1 hr at room temperature, followed by incubation with primary antibodies at 4°C overnight (the primary antibodies were rabbit anti-GAPDH antibody, 1:2000, Cell Signaling, Cat#5174; rabbit anti-p62 antibody, 1:1000, Cell Signaling, Cat#23214; rabbit anti-Tom20 antibody, 1:1000, Cell Signaling, Cat#sc-42406; rabbit anti-LC3A/B antibody, 1:1000, Abcam, Cat#ab128025; rabbit anti-PINK1 antibody, 1:1000, Cell Signaling, Cat#6946; mouse anti-Parkin antibody, 1:1000, Santa Cruz, Cat#sc-32282). The secondary antibodies were incubated for 1 hr at room temperature, then the membrane signals were visualized by a chemiluminescent HRP substrate reagent (Bio-Rad Laboratories, Hercules, CA), and images were captured with Tanon5200 Imaging System (Biotanon, Shanghai, China). The band intensity was assessed with ImageJ software and normalized to that of GAPDH.

Oocytes were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature, permeabilized with 0.5% Triton X-100 for 20 min, then blocked with 1% BSA in PBS for 1 hr at room temperature. The oocytes were incubated with primary antibodies (Alexa Fluor 488 Conjugate anti-α-tubulin monoclonal antibody, 1:200, Cell Signaling, Cat#35652; rabbit anti-Tom20 antibody, 1:100, Cell Signaling, Cat#sc-42406; mouse anti-Parkin antibody, 1:100, Santa Cruz, Cat#sc-32282) at 4°C overnight, and then the oocytes were extensively washed with wash buffer (0.1% Tween 20 in PBS), probed with Alexa Fluor 488 goat anti-rabbit IgG (1:200, Thermo Fisher Scientific, A21206) or Alexa Fluor 594 donkey anti-mouse IgG (1:200, Abcam, ab150108) in a dark room for 1 hr at room temperature. Then oocytes were counterstained with DAPI (10 μg/mL) at room temperature for 10 min. Finally, samples were mounted on glass slides and viewed under the confocal microscope (Nikon A1R-si).