Ab76494

For immunofluorescence staining for synapsin I and II, the sections of the animals (n = 3) were incubated with a rabbit anti-synapsin I (1:50, Abcam #ab64581) or a rabbit anti-synapsin II (1:50, Abcam #ab76494) overnight at room temperature. Then, the sections were incubated with a Cy3-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature. Immunofluorescence images were captured with an Axio Imager 2 microscope (Carl Zeiss). The densities of Synapsin I/II-immunoreactive structures were evaluated on the basis of OD, which was obtained after the transformation of the mean red level using the formula: OD = log (256/mean red level). The OD of background was taken from areas adjacent to the measured area. After the background density was subtracted, a ratio of the optical density of image file was calibrated as % (relative optical density, ROD) and analyzed using NIH Image 1.59 software. A ratio of the ROD was calibrated as %, with control-group designated as 100%.

The mitochondrial protein sample was mixed with the loading buffer and heated at 100 ℃ for 8 min, then separated by SDS-PAGE (Tris-HEPES-SDS gradient 4 to 20% gels) and transferred to a PVDF membrane. The membrane containing the transferred protein was blocked with 5% skim milk (150 mM NaCl, 10 mM Tris, 0.1% Tween-20, pH 8.0) in TBST. The blocked membrane was incubated with anti-β-actin (1:1000, Santa Cruz, CA, sc-47778), anti-histone 3 (1:1000, Abcam, ab1791), anti-VDAC1 (1:2000, Abcam, ab154856), anti-caspase-3 (1:1000, CST, 9665S), anti-cleaved caspase-3 (1:1000, CST, 9664L), anti-PURA (1:1000, Abcam, ab84928), anti-PSPC1 (1:1000, Abcam, ab184123), anti-Dynamin-1 (1:2000, Abcam, ab40758), anti-SYN2 (1:5000, Abcam, ab76494), anti-cytochrome c oxidase subunit (1:2000, Abcam, ab150422), anti-Complex I (1:1000, Abcam, ab110245), anti-beta tubulind (1:1000, Abcam, ab6046) in TBST buffer 4 ℃ overnight. After washing in TBST, membranes were incubated with a 1:3000 dilution of anti-rabbit or anti-mouse IgG HRP secondary antibody diluted in TBST for 1 h. Subsequently, the membrane was washed in TBST (4 × 10 min) and developed using a chemiluminescent reagent from an ECL kit (Thermo Scientific Pierce ECL, USA). Blots were detected on a phosphor imager and analyzed using ImageQuant 350 software.