Quantifying NFIX+ and GFP+ Cells in BNSTp

Brains were dissected from perfused P14 Esr1^Cre/+;Sun1–GFP^lx/+ animals and cryosectioned at 40 μm before immunostaining with primary antibodies to GFP (1:1,000, Aves GFP-1020) and Nfix (1:1,000, Thermo Fisher PA5-30897), and secondary antibodies against chicken (1:300, Jackson Immuno 703-545-155) and rabbit (1:800, Jackson Immuno 711-165-152), as previously described^{16 (link)}. A Zeiss Axioimager M2 System equipped with MBF Neurolucida Software was used to take 20× wide-field image stacks spanning the BNSTp (five sections, both sides). The number of Nfix⁺, GFP⁺ and Nfix⁺GFP⁺ cells was quantified using Fiji/ImageJ from the centre three optical slices by an investigator blinded to condition.