Odyssey clx infrared image system

The effects of drug treatments on striatal DAT and TH levels, highly specific markers for striatal DA nerve endings, were determined by immunoblotting as an index of toxicity. Striatal tissue was dissected from the brain after treatment and stored at −80 °C. Frozen tissue was disrupted by sonication in 1% SDS at 95 °C and insoluble material was removed by centrifugation. The concentration of soluble protein was determined by the bicinchoninic acid method and equal amounts of protein (70 μg/lane) were resolved by SDS-polyacrylamide gel electrophoresis and then electroblotted to nitrocellulose. Blots were blocked in Odyssey blocking buffer (PBS) for 1 h at room temperature. Primary antibodies against DAT (1:1000), TH (1:1000), or GAPDH (1:10,000) were added to blots and allowed to incubate overnight at 4 °C. Blots were washed 3x in Tris-buffered saline to remove unreacted antibodies and then incubated with IRDye secondary antibodies (1:4000) for 1 h at room temperature. Immunoreactive bands were visualized by enhanced fluorescence and the relative densities of TH-, DAT-, and GAPDH-reactive bands were determined by imaging with an Odyssey CLx Infrared Image System (LiCor Biosciences, Lincoln, NE) and quantified using ImageJ software (NIH). DAT and TH relative densities were normalized to the GAPDH level for each lane to control for loading error.