Single bolus ICV injection in neonate mice

Lyophilized ASO was reconstituted in 1× PBS (Thermo Fisher Scientific, 10010023), and the concentration of ASO solution was determined by OD_260nm absorbance. For dosing solutions, reconstituted ASO was diluted to the desired concentration in PBS with 0.01% (weight to volume) fast green dye (Sigma-Aldrich, F7252-5G). PBS with 0.01% fast green dye was prepared as the vehicle control. For ICV injection in P2 or P14 mice, pups were immobilized by gentle restraint on a soft tissue padded surface with two fingers. A 33-gauge needle (0.375 inch long, point style 4, 12° beveled; Hamilton, 7803-05) attached to a 5-μl microvolume syringe (Hamilton, 7634-01) was used for the injection. The coordinates of the injection were about 1 mm lateral from the bregma and −2 mm ventral. Two microliters (for the 20-μg dose) or 3 μl (for the 60-μg dose) of ASO or PBS was injected slowly into one cerebral lateral ventricle. Injected mice were quickly returned to the nest and observed daily for survival and signs of stress. Investigators performing the injections were blinded to study material and genotype.