Anti vdac1

Proteins were separated by SDS–PAGE under reducing conditions and transferred to poly(vinylidene fluoride) membranes. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline with 0.1% Tween 20 (Sigma-Aldrich, P9416; PBST). The blots were incubated overnight at 4 °C with primary antibodies. Immunoblot analysis was performed using an enhanced chemiluminescence procedure (GE Healthcare, RPN2106), and western blot images were captured using the ChemiDoc™MP system (Bio-Rad), Image Lab version 6.1.0. Densitometric analysis was performed using ImageJ software (National Institutes of Health). The following antibodies were used: Cell Signaling Technology: anti-COXIV (3E11, #4850), 1:1000; anti-VDAC1 (D73D12, #4661), 1:1000; anti-Tomm20 (D8T4N, #42406), 1:1000; anti-GAPDH (14C10, #2118), 1:10000; anti-Cytochrome c (136F3, #4280), 1:1000; anti-LC3B (#2775), 1:1000; Novus Biologicals: anti-Dhps (NBP1-82648), 1:1000; BD Biosciences: anti-Eif5a (611976), 1:1000; Sigma-Aldrich: anti-Dohh (HPA041953), 1:1000; Millipore: anti-Hypusine (ABS1064), 1:1000; anti-puromycin (12D10, #MABE343), 1:1000; Abcam: anti-Tfam (ab131607), 1:1000; anti-PGC1α (ab54481), 1:1000; anti-TFEB (ab2636), 1:1000; and Atp5a, Uqcrc2, Sdhb, and Ndufb8 were detected using Total OXPHOS Rodent WB Antibody Cocktail (ab110413), 1:1000. Primary antibodies were diluted in 1% BSA containing PBST.

Brain sections were embedded in Tissue-Tek (OCT compound, Fisher Scientific) and immediately frozen at −80 °C. Then, sections of frozen brain (10 µm thick) were performed using a cryostat (Leica Microsystems) and were mounted on glass slides, and air dried. After a step of fixation with 4% of paraformaldehyde and of permeabilization with a mixture of PBS/0.2% triton X-100, nonspecific binding was blocked with PBS containing 0.1% bovine serum albumin (BSA, Sigma Aldrich), 5% non-immune goat serum (NGS) and 0.05% tween 20 for 20 min at room temperature. Then, the sections were incubated at 4 °C overnight in the same solution supplemented with primary antibody, anti-Voltage-dependent anion-selective channel 1 (anti-VDAC1, Novus biologicals, Littleton, CO). After 3 washes with PBS, sections were incubated with FITC-conjugated secondary Antibodies (LifeTechnologies) for 1h30 at room temperature in the dark. After 3 washes with PBS, cover-slips were mounted with a drop of mounting medium for fluorescence with DAPI (Vector Laboratories, Peterborough, UK). After immunohistochemistry, microphotographs were acquired with DM5500 B fluorescence microscope equipped with a Photometrics CoolSNAP HQ2 camera (Leica) for detection of DAPI or FITC with 10× and 63× objectives.