Visualizing Mitochondrial Morphology in Mouse Brain

Two methods be used for observing mitochondrial morphology in mice brain. A) injection AAV-BBB-Mito DsRed by the tail vein 100 μl/mice. After 4 weeks, mice were anesthetized with pentobarbital sodium (80 mg/ml) and perfused with ice-cold 0.9% saline, finally embedded in OCT (SAKURA#4583). Samples were frozen by liquid nitrogen & isopentane for 45 s and then cut into 100 μm slices. B) Immunostaining. Mice was anesthetized with pentobarbital sodium (80 mg/ml) and perfused with ice-cold 0.9% saline, finally embedded in OCT (SAKURA#4583). Samples were frozen by liquid nitrogen & isopentane for 45 s and then cut into 8 μm slices. The slices fixed and permeabilized by acetone for 5 min, followed by blocking with 5% donkey serum in PBS with 0.2% BSA for 1 h at room temperature. Incubate with primary antibody (VDAC1, ab14734, abcam, 1:250) in blocking solution overnight at 4 °C, then wash with PBST (0.2% Triton X-100) 3 times for 5 min. Secondary antibody (1:1000) was used for 4 h at room temperature under darkness. Images were acquired using Leica SP8 laser scanning confocal microscope equipped with a 63 NA oil objective as Z stacks, with identical imaging parameters among different phenotypes in a blinded fashion. The length and area of mitochondrion were measured by Image J (Fiji)^{51 (link)}.