Ab254257

Fetal brain was collected (e19) and stored in 4% PFA solution (paraformaldehyde) for 7 days, transferred to PBS 0.1M 10% sucrose solution with a gradual increase to PBS 30% solution overnight, frozen in OCT compound (Fisher HealthCare, Houston, TX, USA), and sectioned (15 µm coronal sections). Antigenic retrieval was performed on slices using Citrate-EDTA buffer for 40 min at 90 °C. Sections were blocked in 5% albumin (0.1M PBS + 0.25% Triton X-100) for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. The antibodies used were: NPY (1:100 #11976S, Cell Signaling© Danvers, MA, USA), POMC (1:50 ab254257, Abcam, Cambridge, MA, USA), and NEUN (1:500 ab104224, Abcam, Cambridge, MA, USA). Sections were incubated for 90 min with secondary antibodies Alexa 488 Rabbit (1:200, Invitrogen, Ref. Z25302), Alexa 568 mouse (1:200, Invitrogen, Ref. A11004). DAPI (1:1000 Life Technologies, Carlsbad, CA, USA) was used for nuclear labeling. Sections were visualized at 40X lens, captured using Upright LSM780-NLO Microscope (National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC), University of Campinas), and quantified using ImageJ software (version 1.53 k, http://rsbweb.nih.gov/ij/ accessed on 11 August 2021). Cell counting was performed on 3 sections from the same fetus and 3 mice per group.