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To generate a riboprobe for Avp mRNA, Avp cDNA (cds, 21-495) was amplified by RT-PCR using mouse brain cDNA as a template (and with the following primers: Avp forward, GGGAAGCTTCACTACGCTCTCCGCTTGTT; Avp reverse, GGGTCTAGAGGGCTTGGCAGAATCCA). FISH signal of the fluorescein-labeled dnBMAL1-specific riboprobe was detected with anti-fluorescein horseradish peroxidase (HRP) conjugate (PerkinElmer, Boston, MA, USA) and amplified with a cyanine-3 substrate kit (Cy3 TSA Plus; PerkinElmer). Data h as been generated by GSA Content Generator Dem over sion! After quenching with H2O2, FISH signal of the digoxigenin-labeled Avp riboprobe was detected with anti-digoxigenin-POD, Fab fragments (Roche) and amplified with a cyanine-5 substrate kit (Cy5 TSA Plus, PerkinElmer). 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Behavioral testing started 16 days after the beginning of the injections and lasted 8 days with tests performed in the following order: d0, first injection; d15, food deprivation; d16, NSFT and HCF; d18, OFT; d19, EPM; d25, staggered sacrifice 4 h after last morning injection. The first experiment was conducted to study the baseline effects of HFD; 16 CD and 16 HFD rats were used for western blot analysis after 8 or 16 weeks; another 16 CD and 16 HFD rats were used for molecular, biochemical, and behavior studies and killed after 16 weeks; and 8 CD and 8 HFD rats were used for glucose tolerance tests. Behavior was video recorded and exploratory behavior was determined. Sucrose and water consumption were determined by measuring the change in the volume of fluid consumed. Sucrose preference was defined as the ratio of the volume of sucrose versus total volume of sucrose plus water consumed during the 1 h test. Rats were exposed to a palatable sucrose solution (1%; Sigma) for 96 h. 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