Quantifying Cerebellar Gene Expression

Radiochemical in situ hybridization was performed on coronal 12 μm cryosections of the cerebellum. DNA probes were labeled with ³⁵S-dATP (Perkin Elmer) and separate sections were hybridized for each gene as previously described (Klitten et al., 2008 (link)). Probe sequences for detection of period 2 (Per2) and nuclear receptor subfamily 1 group D member 1 (Nr1d1) transcripts have been previously published (Rath et al., 2012 (link)). Hybridized sections were exposed to an X-ray film and the autoradiographical images were digitized and quantified using Scion Image Beta 4.0.2 (Scion). Optical densities of the hybridization signal were converted to dpm/mg by use of a standard curve of known values of ¹⁴C standards co-exposed on every X-ray film. For each animal, 4 tissue sections were measured and averaged. The area of densitometric measurement was confined to the granular layer and Purkinje cells of a dorsal folium of the vermis.