rformed by lightly boiling sections in 10 mM sodium citrate, 0.05% Tween-20, pH 6.0 for 20 min, followed by cooling at RT for 30 min. Sections were blocked with 5% FCS, 0.1% Triton X-100 in PBS and primary antibody diluted to 1% FCS in PBS was incubated TAK 438 free base pubmed ID:http://www.ncbi.nlm.nih.gov/pubmed/19636622 overnight. Cultured cerebellar granule cells growing on coverslips were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 
in PBS for 10 min. Coverslips were blocked with 10% FCS in PBS and primary antibody diluted to 1% FCS in PBS was incubated overnight. Secondary antibodies were diluted to 1% FCS in PBS and incubated for 30 min. Nuclei were counter stained with 1 ng/ml Hoechst 33258. For stereological analysis of interneurons, mice were euthanized at P14 and P30. Brains were removed, bisected along the midline and immersion fixed in 4% paraformaldehyde for one week before cryoprotection in 30% sucrose in TBS containing 0.05% sodium azide. Frozen sagittal sections of 40 mm were cut through the brains and collected as series in cryoprotectant solution. One-in-six series of freefloating sections were immunostained with anti-parvalbumin or Nissl stained as described previously. The specificity of all immunostainings was verified by controls in which the primary antibody was omitted. Antibodies for immunohistochemistry Mouse anti-carp parvalbumin ; rabbit-anti-GABA ; rabbit anti-human synapsin 1 ; mouse anti-rat vesicular GABA transporter, rabbit anti-rat gephyrin, rabbit anti-rat GABRA6, rabbit anti-rat vesicular glutamate transporters 1 and 2 . Secondary antibodies were as follows: anti-mouse-Alexa Fluor PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639555 564, anti-rabbit-Alexa Fluor 564, anti-mouse-Alexa Fluor 488, anti-rabbit-Alexa Fluor 488 . Counts of interneuron number StereoInvestigator software was used to obtain unbiased optical fractionator estimates of interneuron number in molecular layer of cerebellum from parvalbumin and Nissl stained 40 mm sections. Parvalbumin was used for P14 as at this age molecular layer still contains migrating granule cells that are difficult to reliably differentiate from interneurons in Nissl stain. All neuronal cell bodies on molecular layer of P30 mice were considered to be interneurons in Nissl stained sections. Measurements were performed as described previously. The cut section thickness was nominally 40 mm, but after mounting and dehydration the actual thickness of sections was 15 mm. The optical fractionator was run using a guard height of 0.5 mm at both the top and bottom of the section, meaning that the actual depth of the fractionator was 14 mm. The following sampling scheme was used: grid area 30 625 mm2, frame area 2 400 mm2. The data were expressed as mean neuron number 6 standard error. The unpaired Student’s t-test was used for statistical analysis the mice were immersiofixed Gene Expression Alterations in Cstb2/2 Mouse 5.0c; GraphPad Software, La Jolla, CA, USA)) with p,0.05 considered as statistically significant. The mean coefficient of error for all individual optical fractionator estimates was calculated according to the method of Gundersen and Jensen and was less than 0.07 in all of the analyses. Results Altered expression of synaptic genes in Cstb2/2 mouse cerebellum at postnatal day 7 The genomewide microarray analysis of P7 cerebella from Cstb2/2 mice revealed 82 differentially expressed probe sets within 61 genes, of which 30 probe sets were upregulated and 52 were downregulated. The over-represented GO categories are summarized in Analysis of
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