Immunohistochemical Analysis of c-Fos and OXT

Serial 40-μm-thick sections were rinsed twice with 0.1 M phosphate-buffered saline (PBS) and washed in 0.1 M Tris buffer (pH, 7.6) containing 0.3% Triton X-100. Sections were incubated for 72 h at 4 °C in primary antibody solution (goat anti-c-Fos, Santa Cruz Biotechnology, TX, USA; 1:500 or rabbit anti-OXT, Sigma-Aldrich, MO, USA; 1:5000)^{65 (link)}. After washing twice in 0.3% Triton X-100 in PBS, floating sections were incubated for 24 h at 4 °C with a secondary antibody (Alexa Fluor 546 donkey anti-goat IgG or Alexa Fluor 488 donkey anti-rabbit IgG; Molecular Probes, OR, USA; 1:2,000 in PBS containing 0.3% Triton X-100)^{65 (link)}. Sections were washed twice in PBS and then mounted on the slides and coverslipped using vectashield (Vector Laboratories Co. Ltd., CA, USA)^{66 (link)}. Images of Fos⁺, OXT⁺, and Fos⁺/OXT⁺ double-labelled cells were counted manually by two researchers who were blinded to avoid bias. The number and percentage of Fos⁺, OXT⁺, and Fos⁺/OXT⁺ cells in the SON and PVN were estimated.