Current responses were recorded using

an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) and the pclamp software (version 9.2; Molecular Devices). Signals were filtered at 1 kHz and digitized at 4 kHz. 4-Hydroxytamoxifen (4OHT; Sigma) was dissolved in ethanol at a concentration of 20 mg/mL and diluted with 9 volumes of corn oil (Sigma). The diluted 4OHT (200 μL per mouse) was intraperitoneally injected into mice at P6. Under deep anesthesia, the mice were fixed by cardiac perfusion with 0.1 m sodium phosphate buffer (PB), pH 7.4, containing 4% paraformaldehyde (4% PFA/PB); the cerebellum was then removed and SCH727965 datasheet soaked in 4% PFA/PB for 4–24 h. After rinsing the specimens with PBS, parasagittal slices (50–100 μm thick) were prepared using a microslicer (DTK-2000; Dosaka, Kyoto, Japan) and subjected to immunohistochemical staining with the following antibodies: guinea pig anti-calbindin (1 mg/mL; Nakagawa et al., 1998), rabbit anti-calbindin (1 : 500; Millipore, Bedford, MA, USA), mouse anti-NF-H (1 : 1000;

Covance, Berkeley, CA, USA), guinea pig anti-glial fibrillary acidic protein (GFAP; 1 mg/mL, provided by Dr Watanabe at Hokkaido University), guinea pig anti-vesicular glutamate transporter VGluT1 (1 μg/mL; Miyazaki et al., 2003), mouse anti-HA (1 : 500; Covance) and goat anti-RORα (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). PD0332991 clinical trial Sections were permeabilized with 0.1 or 0.2% Triton X-100 in PBS, blocked with 10% donkey serum in PBS, and incubated overnight with primary antibodies followed by 1–2 h of incubation

with Alexa Fluor- (Invitrogen, Carlsbad, CA, USA) or DyLight- (Jackson Immunoresearch Laboratory, West Grove, PA, USA) conjugated secondary antibodies. For fluorescence Nissl staining, the sections were incubated with NeuroTrace Red (1 : 100; Invitrogen) for 1 h. The stained slices were viewed using a confocal laser-scanning microscope (Fluoview; Olympus, Tokyo, Japan or LSM710; Carl Zeiss, Göttingen, Germany). To determine the cell-type specificity, parasagittal slices of cerebellar vermis (50 or 100 μm thick) were immunostained for calbindin, and the numbers of EGFP-positive cells that were calbindin-immunopositive or -immunonegative in the cerebellum were counted. Statistical significance was defined by the χ2 test with Bonferroni correction. To separate the emission fluorescence of Mito-ECFP, EGFP-β-actin and Carnitine palmitoyltransferase II DsRed2, z-stack images of spectral data were obtained from a cerebellar slice by confocal microscopy (LSM710; Carl Zeiss). The images were processed by a linear unmixing algorithm (Zimmermann et al., 2003) to generate three-fluorescence images. The reference spectral data were obtained from human embryonic kidney 293 cells expressing only one of the three fluorescent proteins (Mito-ECFP, EGFP-β-actin and DsRed2). To study the effect of RORα1DN-HA on Purkinje cell development, parasagittal slices of cerebellar vermis (100 μm thick) were immunostained for calbindin and HA.