Sheep anti α msh

Sections were processed for immunofluorescence using standard procedures (Bouret et al., 2004b (link); Steculorum et al., 2015 (link); Croizier et al., 2016 (link)). The primary antibodies used for IHC were as follows: sheep anti-α-MSH (1:40,000, Millipore; Steculorum et al., 2015 (link)), sheep anti-NPY (1:3,000, Chemicon; Lee et al., 2013 (link)), rabbit anti-β-endorphin (1:5,000, Millipore; Grayson et al., 2010 (link)), rabbit anti-POMC (1:2,000, Phoenix Pharmaceuticals; MacKay et al., 2017 (link)). The primary antibodies were visualized with Alexa Fluor 568 goat anti-rabbit IgGs or Alexa Fluor 568 donkey anti-sheep IgGs (1:200, Invitrogen). Sections were counterstained using bisbenzamide (1:10,000, Invitrogen), to visualize cell nuclei, and coverslipped with buffered glycerol (pH 8.5).

Three-week-old brains derived from 10 different litters (n = 5 litters/maternal treatment) were dissected and fixed in 4% paraformaldehyde overnight at 4°C. Cryoprotected 3-week-old brains were frozen in smashed dry ice and sectioned using a cryostat (Leica CM 1950). Selected 20 μm-thick sections (1 out of 4 sections) throughout the PVH were used. For α-MSH staining, sections were blocked with 2% donkey serum in KPBS + 0.4% Triton X-100 for 1 h and subsequently incubated with sheep anti-α-MSH (1:750; Millipore) in blocking solution overnight at 4°C. As secondary antibody, a donkey anti-sheep Alexa Fluor 488 or 594 (1:300; Life Technologies) in KPBS + 0.4% Triton X-100 was used (2 h at room temperature). For AgRP staining, sections were blocked with 2% chicken serum in KPBS + 0.4% Triton X-100 for 1 h and subsequently incubated with rabbit anti-AgRP (1:500; Phoenix Pharmaceuticals) in blocking solution for 48 h at 4°C. As secondary antibody, a chicken anti-rabbit Alexa Fluor 488 (1:300; Life Technologies) in KPBS + 0.4% Triton X-100 was used (2 h at room temperature).