248 Fig. 1 Generation of condition- al Cacna1a Purkinje cell- specific knockout mice. a Sche- matic representation of the ge- nomic structure of the relevant part of the floxed and recom- bined Cacna1a alleles. Black boxes indicate exons (E). Trian- gles indicate the position of loxP sites. Arrows indicate the posi- tion of the primers used for the RT-PCR. b RT-PCR of cerebellar RNA amplifies a 580-bp prod- uct from the non-recombined Cacna1a transcript and a 490-bp product from transcripts that lack exon 4. c–f Anti-CaV2.1- α1A immunostaining in cerebel- lar sections of P30 WT (c, e) and PCα1KO (d, f) mice. CN cerebellar nuclei, G granule cell layer, M molecular layer, PC Purkinje cell layer, WM white matter. Roman numbers indicate the cerebellar lobules Cerebellum (2012) 11:246–258 quently, the sheet of dura mater separating cerebrum and cerebellum was removed, tissue was embedded in 10% sucrose in 10% gelatine, and the gelatin was fixed with 30% sucrose in 10% PFA for 2.5 h at room temperature. This was followed by an overnight incubation in 30% sucrose in 0.1 M phosphate buffer at 4°C. Forty-micrometer sagittal sections were made using a freezing cryotome (Leica, Bensheim, Germany). Part of the sections was processed for silver staining, which selectively stains dying neurons [23]. The remainder of the sections was processed directly for free- floating immunohistochemistry. Sections were incubated in 10% heat-inactivated normal horse serum (NHS) with 0.5% Triton-X100 in TBS, for 2 h and then incubated either with primary rabbit polyclonal anti-CaV2.1-α1A antibody (ACC-001, 1:100; Alomone Labs, Jerusalem, Israel), or with rabbit polyclonal anti-Calbindin antibody (CB38, 1:10,000; SWANT, Bellinzona, Switzerland) dilut- ed in 1% NHS with 2% heat-inactivated fetal calf serum and 0.4% Triton-X100 in TBS at 4°C. Next, sections were washed and incubated for 2 h at room temperature with a secondary biotin-labeled goat anti-rabbit antibody (1:200; Vector Laboratories, Burlingame, CA). For protein detec- tion, sections were incubated with the avidin-biotin- peroxidase complex (ABC kit, 1:100, Vector Laboratories) for 2 h at room temperature, and developed in 0.1 mg/ml diaminobenzidine in 0.005% H2O2. Sections were Nissl stained, air dried, and cover slipped. Paraffin-embedded cerebellar sections (5 μm) were processed for standard haematoxylin and eosin (HE) staining. Golgi-Cox Staining Brains from 30-day-old mice (two PCα1KO and two WT) were isolated after decapitation and processed for Golgi impregnation. Brains were rinsed briefly in Milli-Q water. Tissue was further processed for Golgi-Cox staining using FD Rapid GolgiStain™ Kit (MTR Scientific, Ijamsville, MD) with 120-μm thick sections. Light microscopy images were used to assess the dendritic arborisation of Purkinje cells using the linear Sholl analysis [24–26]. In short, 7 to