Anti darpp32

SD rats (Slc:SD; RRID: RGD_12910483) were obtained from NIPPON SLC. All efforts were made to minimize animal suffering and the number of animals used for the study. Animals were housed, handled, and bred according to the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. Recombinant human BDNF was kindly provided by Sumitomo Dainippon Pharma and was dissolved in a 2% bovine serum albumin (BSA)/phosphate‐buffered saline (PBS). The final solvent concentration in the culture medium was 0.1%. Anti‐microtubule‐associated protein‐2 (anti‐MAP2; 1:400; Sigma‐Aldrich), anti‐dopamine‐ and cAMP‐regulated phosphoprotein, 32 kDa (anti‐DARPP‐32; 1:30; Santa Cruz Biotechnology), and anti‐glial fibrillary acidic protein (GFAP; 1:400; Merck Millipore) for immunocytochemistry were used. Anti‐rabbit and anti‐mouse IgG secondary antibodies conjugated to Alexa Fluor 488 (1:400) or 546 (1:400) were purchased from Molecular Probes.

Free-floating sections were subjected to pretreatment with 0.3% hydrogen peroxide for 1 h, washed three times in PBS, blocked in PBS containing 4.5% NGS for 1 h, then incubated overnight at 4°C in PBS containing 3% NGS, 0.2% Triton™ X-100 and one of the following antibodies: anti-DARPP32 (1:1000, rabbit, Santa Cruz Biotechnology), anti-NeuN (1:2000, mouse, Millipore), anti-ubiquitin (1:1000, rabbit, Wako Chemicals), or anti-HA (1:500, mouse, clone 11 Covance). Sections were rinsed three times in PBS and then incubated with the appropriate anti-IgG biotinylated antibody (Vector Laboratories) at a dilution of 1:5000, for 1 h. Staining was visualized by adding avidin-biotinylated peroxidase and incubating with DAB or VIP substrate (Vector Laboratories) for 1 min. For NeuN immunostaining, we used the MOM immunodetection kit (Vector Laboratories). Stained sections were mounted on microscopy slides.