Fluoromount g containing 4 6 diamidino 2 phenylindole dapi

All mice were overdosed with Fatal-plus (0.01 ml/g; Vortech Pharmaceutical, Ltd.) and perfused with 10 ml of cold saline followed by 15 ml of 10% PB formalin. Brains were stored in 10% formalin/30% sucrose (4°C) before sectioning.
All brains were frozen and alternate coronal sections were obtained with a sliding microtome at 40-μm thickness and stored floating in TBS with 0.03% sodium azide. Sections containing the OB, AON, anterior PCX (aPCX), and/or OT were rinsed in deionized water and mounted on slides using Fluoromount-G containing 4',6-diamidino-2-phenylindole (DAPI; Invitrogen). We selected 4–13 sections of each brain region for quantification, ensuring that sections spanned the anterior-posterior length of each region.

Animals were deeply anesthetized and transcardially perfused with PBS, followed by PBS supplemented with 4% paraformaldehyde. Brains were sectioned on a cryostat (Cryostar NX-70) at 40 μm and stored in cryoprotectant buffer at −20°C until use. Sections were thawed, washed in PBS, and blocked and permeabilized in PBS supplemented with 0.1% Tween (PBST) and 5% normal donkey serum for 1 hour at RT. Next, sections were incubated in PBST supplemented with 0.5% rabbit anti-NeuN (1:1000, AB104225, Abcam, UK) at 4°C overnight. After PBS washes, sections were incubated in anti-rabbit Alexa Fluor 568 antibodies (1:500, Molecular Probes, OR, USA) for 1 hour. Sections were mounted and coverslipped in Fluoromount-G containing 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). Z-stacks with ×60 magnification of eGFP-injected regions and the corresponding sham-injected contralateral region were imaged using a Keyence microscope. Z-stacks were projected with maximal intensity, and NeuN-positive cells were manually counted in ImageJ by a blinded observer.