Following standard perfusions as described in [18 (link)], 0.1 mm sections were cut on a vibratome. Sections were blocked in PBS with 0.3% Triton X-100 and 1% bovine serum albumin (blocking solution) for 1 h, incubated with goat anti-orexin (1:1000; Cat# 8072, RRID: AB_653601, Santa Cruz) over-night, washed, incubated with Alexa 647 conjugated donkey anti-goat (1:1000; Cat# A-21447, RRID: AB_2535864, Invitrogen/Thermo Fisher Scientific) for 3.5 h, washed and mounted. Antibodies were applied in blocking solution. Confocal micrographs were acquired on a Nikon A1 and merged in imageJ.