T-4092

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Publication protocol

Immunohistochemistry (IHC) and in situ hybridization (ISH) were performed on 30-μm coronal sections as previously described (18). Rabbit anti-peptide YY (Peninsula Laboratories, San Carlos, CA) and rabbit anti-5HT (Immunostar, Wisconsin) antibodies were used to detect PYY and serotonin [5-hydroxytryptamine (5-HT)] neurons. DIG-labeled ISH riboprobe was generated using Pyy mouse sequence. Imaging was performed with an Olympus BX51 microscope combined with SimplePCI software. Confocal microscopy was performed on a Bio-Rad MRC1000 microscope. For phospho-signal transducer and activator of transcription 3 (pSTAT3) immunostaining, 24-h-fasted 3-mo-old PYY-Cre;ROSA-EYFP mice were injected with 5 μg/kg ip recombinant mouse leptin (R&D Systems, Abingdon, UK) or vehicle and perfused 45 min later. Brain stem and hypothalamus sections were stained for pSTAT3 as previously described (54, 54) using rabbit pSTAT3 (Tyr705) antibody (Cell Signaling Technology, UK).

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