Gt15023

Mice were anesthetized by i.p. injection of an overdose of pentobarbital, and then transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) in PBS. After post fixation overnight, the brain was isolated and cryoprotected with 30% sucrose for 2 days. For further imaging and analysis, whole brains were coated with tissue freezing medium and coronal sections (40 μm thick) were prepared on a cryostat (Leica CM1900). Some mouse brains were cut sagittally to better visualize the axon projections. Brain sections mounted on chrome-gelatin subbed slides were washed with PBS four times every 6 min. For immunofluorescent staining, the sections were blocked with 3% BSA in PBS-0.3% Triton X-100 and subsequently incubated with primary antibodies rabbit anti-POMC (1:200, catalog# H-029-30, Phoenix Pharmaceuticals; overnight), goat anti-AgRP (15 μg/ml, catalog# GT15023, Neuromics; 72 h), or chicken anti-GFP (1:500, catalog#ab290, Abcam; overnight) at 4°C. Sections were then incubated with secondary antibodies, Alexa647 donkey anti-rabbit (1:500, Jackson ImmunoResearch), Alexa647 donkey anti-goat (1:500, Jackson ImmunoResearch) or Cy2 anti-rabbit (1:500, Jackson ImmunoResearch) for 4 h at room temperature. Finally, these brain sections were cover-slipped with 50% DAPI-glycerol mounting medium.