Goat anti chicken alexa488

Pomc-Cre and AgRP-Cre mice were anesthetized with ketamine-xylazine and perfused intracardially with saline (0.9% NaCl) followed by 10% buffered formalin. Fixed brains were immersed in 30% sucrose and 0.01% sodium azide in PBS at 4 °C for 2 days. Next, 3 sets of coronal sections (40-μm-thick) were cut in a freezing microtome Leica CM1850 UV and stored at −20 °C in cryo-protectant.
One set of free-floating sections from each animal was washed three times in TBS 0.1 M for 10 min each and incubated in blocking solution (2% donkey serum + 0.3% Triton X-100) in TBS 0.1 M for 60 min. Then, sections were incubated in rabbit anti-POMC (1/200; Phoenix pharmaceuticals, Cat# H-029-30), goat anti-AgRP (1/5000; Phoenix pharmaceuticals, Cat# H-003-57) or chicken anti-GFP (1/1000, Invitrogen A10262 Cat# 10,524,234) in blocking solution for 24 h at 4 °C. After incubation in the primary antibody, sections were rinsed with TBS 0.1 M three times for 10 min each and then incubated in the secondary antibody: Cy3 donkey anti-rabbit (Jackson ImmunoResearch Labs Cat#711-165-162); Cy3 donkey anti-goat (Jackson ImmunoResearch Labs Cat#705-165-147) and goat anti-chicken Alexa 488 (abcam Cat# ab150169) for 60 min at room temperature. Sections were then washed and coverslipped with Fluorogel coverslip mounting solution [27 (link),28 (link)].