Water Maze Behavioral Testing

“Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine which of these antibodies displayed the most specific binding to human α-syn, of these antibodies, 9E4 displayed the most specificity”

“A total of 40 α-syn tg mice (6 m/o, n = 20 mice per group) received weekly intraperitoneal (IP) injections (10 mg/kg) for 6 months with the CT-α-syn antibody (9E4) and IgG1 control”

“An additional group of non-tg mice treated with the 9E4 antibody (n = 12) and the IgG1 control (n = 12) was included as control for behavioral and neuropathological studies”

“Mice were bled once a month and antibody titers monitored by enzyme-linked immunosorbent assay (ELISA)”

“At the end of the studies, mice were tested for functional effects in the water maze”

“the mouse monoclonal antibody 9E4 was concentrated with a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA) and linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions”

“non-tg (total n = 18) and α-syn tg mice (total n = 18) (6 m/o) were injected intravenously (IV) with the 9E4-FITC or a non-immune control FITC tagged IgG1 at a concentration of 1 mg/kg”

“Mice were sacrificed 3, 14 and 30 days after injection (n = 3 per group). CSF from these mice was used to immunolabel cortical sections from antibody-naive animals”

“non-tg (total n = 8) and α-syn tg mice (total n = 8) (6 m/o) were injected intravenously (IV) with the FITC-labeled β-syn or a non-immune control FITC tagged IgG1 at a concentration of 1 mg/kg”

“Mice were sacrified at 14 days post-injection. CSF from these mice was also used to immunolabel slides from FITC tagged β-syn niave non-tg and α-syn tg mice”

“the right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis, while the left hemibrain was snap-frozen and stored at −70°C for subsequent protein analysis”