35 s datp

Antisense oligoprobes complementary to rat and mouse tph2 mRNA (5′-TCC GTC CAA ATG TTG TCA GGT GGA TCC AGC CTC ACA ATG GTG GTC-3′; position 505; NM_173391) were synthesized by and purchased from Integrated DNA Technologies (Coralville, Iowa). The oligonucleotides were labeled with [35-S]dATP (Perkin Elmer, Waltham, Massachusetts) at the 3′ end using terminal deoxynucleotidyltransferase (Roche, Tucson, AZ) and purified using the QiaQuick Nucleotide Removal kit (Qiagen, Valencia, CA). Each slide was coverslipped with 110 μl of the hybridization buffer containing 0.1 M EDTA, SSC, single-stranded salmon sperm DNA, torula yeast tRNA, Denhardt's solution, formamide, dextran sulfate, and 1M DTT. Sections were incubated in a humidified chamber for 16 h at 42°C. Following hybridization, coverslips were removed and the slides were washed four times in 1X SSC buffer for 15 min at 55°C, once in 0.5X SSC buffer for 20 min at room temperature, and once again in 0.1X SSC buffer for 20 min at room temperature. Slides were then dipped in ddH₂O and air-dried. Slides were exposed to BioMax Kodak autoradiography film in a sealed film cassette in a dark room at room temperature for 4 days.

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.