Anti drp1

Immunoblot was carried out as others described.23 Briefly, the cells were lysed for 20 minutes on ice in RIPA lysis buffer containing a protease inhibitor cocktail and DMSF. The samples were subjected to 12% SDS‐PAGE and transferred to nitrocellulose membranes. Blots were probed with primary antibodies anti‐MARCH5 (Abcom, 1:1000), anti‐Lc3 (Abclone, 1:1000) anti‐KLF4 (Affinity, 1:1000), anti‐Drp1 (Affinity, 1:1000), anti‐Ubiquitin (Affinity, 1:1000) and anti‐β‐actin (TransGen, 1:2000) at 4°C overnight with gently shaking. After three times washing with PBS, the horseradish peroxidase (HRP)‐conjugated secondary antibodies were added. Antigen‐antibody complexes were visualized by enhanced chemiluminescence. Enhanced ECL TM prime detection reagent (GE) used to visualize antigen‐antibody complexes, the density quantified by Image J.

Immunofluorescence staining was carried out on paraffin-embedded brain tissue sections. Briefly, anti-DRP1 (1:100; Affinity Biosciences, OH, USA) or anti-SIRT1 antibodies (1:200; Servicebio, Wuhan, China) were incubated on the sections for 10 h, and the secondary antibody (1:300; Servicebio, Wuhan, China) was then incubated on the sections for 50 min. Lastly, the sections were counterstained with DAPI before observation by a fluorescence microscope. In this study, the mean fluorescence intensity of images was calculated by Image J software (version 1.8.0).