Alexa 647 donkey anti rabbit

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.

Medial and lateral ventricular walls from P10 mice were isolated in cold PBS and sectioned using a Leica VT1000S vibratome (Leica, Wetzlar, Germany). Tissue was fixed in 4% PFA in PBS O/N at 4°C, followed by blocking with 2.5% BSA, 0.2% Triton X-100 in PBS for 4 hours at RT. Primary antibodies used: N-cadherin (rabbit, 1:50; sc-7939, Santa Cruz Biotechnology, Santa Cruz, CA), γ-tubulin (mouse 1:250; ab11316, Abcam, Cambridge, MA). Samples were incubated with primary antibody for 24–48 hours at 4°C. Secondary antibody incubation was performed at 4°C for 48 hours using Alexa 488 goat anti-mouse (1:250, Thermo-Fisher, Waltham, MA) and Alexa 647 donkey anti-rabbit (1:250, Jackson ImmunoResearch, West Grove, PA). Tissues were placed on glass slides, mounted and imaged using an Andor WD Spinning Disk confocal microscope system equipped with an Andor Neo sCMOS camera (Andor, Belfast, UK).