Image gause software

Striatal tissues were collected in cold lysis buffer containing 1% Triton X-100, 1 mM EDTA in phosphate-buffered saline (PBS), protease inhibitor cocktail, homogenized, and centrifuged at 10,000 × g for 10 min at 4°C. Protein concentration was determined using a BCATM protein assay kit (Thermo Fisher Scientific, Waltham, MA, United States) and assessed by Western blotting. Equal aliquots of the samples were denatured at 100°C, separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, United States). Membranes were incubated in a blocking buffer containing 5% BSA in TTBS for 1-h at room temperature. Immunodetection was performed by incubating membrane blots overnight at 4°C separately with the following primary antibodies (1:1000): anti-CRMP-2 (IBL, Gunma, TS, Japan), anti-Cdk5-p35/25, anti-calpastatin, and anti-synapsin-II (Cell Signaling, Dallas, TX, United States). For chemiluminescent detection, membrane blots were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000) for 2-h at room temperature. Data collection and processing of the integrated optical density of the bands were performed with a luminescent image analyzer (LAS-3000) and IMAGE GAUSE software (Fuji Photo Film, Japan).