Imaging and quantification of immunohistochemistry

Images were captured using a confocal Leica TCS SP-8-X microscope, equipped with a ×40/1.30 oil objective with the acquisition software (Leica ASX V.3.5.5.19976). Next, z-stacks were taken with optical sections of 0.9 μm. Laser intensities were kept constant throughout all related conditions. Images were imported into FIJI where maximum intensities were projected. For representative images, adjustments in brightness and contrast for each channel were kept constant throughout all related conditions, whereas for quantifications of POMC and tdTomato signals, all channels were kept unmodified and one to four sections were quantified per mouse and area. Images were converted to 8-bit, and the threshold for signal detection for each channel was determined by visual judgment and consistently applied to all images. ROIs were defined around corresponding anatomical locations and raw integrated densities measured within ROIs for POMC and tdTomato signals.