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Trypsin was neutralized with 500 l complete medium, the cells were resuspended and 100 l of the suspension was mixed with 6.9 ml Beckman Coulter Isoton II diluent (Fisher Scientific, NC2470899) for counting with a Beckman Coulter Z1 Coulter Cell Particle counter

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Formalin-fixed paraffin-embedded brain sections (3 sections per mouse per triplex) underwent RNAscope in situ RNA hybridization (ISH) for the detection of Lepr (ACDBio probe #418858-C2), Glp-1r (ACDBio probe #418858-C2), Gipr (ACDBio probe #319128-C1), Pomc (ACDBio probe #314088-C2) and Agrp (ACDBio probe #400718-C1), followed by immunofluorescence (IF) staining for pSTAT3 protein (Cell Signaling anti-pSTAT3 #9145)

Greg Lopes, a spokesman for the Pharmaceutical Care Management Association, an industry group representing benefit managers, refuted those claims