Bx60 light microscope

Sections were examined using an Olympus BX60 light microscope equipped with fluorescent epi-illumination and a dark-field condenser. Images were captured at 2048 × 2048 pixel resolution with a SPOT Xplorer digital CCD camera (Diagnostic Instruments, Sterling Heights, MI, USA) using 4–40× objectives. Confocal images were acquired with a Nikon Eclipse E800 confocal microscope equipped with a BioRad Radiance 2100 Laser Scanning System using 20–60× objectives at an optical thickness of 1–3 μm. Images were adjusted using the “levels” and “sharpness” commands in Adobe Photoshop CS 8.0. Full resolution of the images was maintained until the final versions, which were adjusted to a resolution of 300 dpi.

One set of every fourth free-floating section from the five rat dams used for single-labeling of galanin was used for double labeling of galanin and oxytocin. First, galanin was immunolabeled using FITC-tyramide amplification immunofluorescence, as described above. Sections were then incubated in citrate buffer at 70 °C for 1 h, and then placed in mouse anti-oxytocin primary antiserum (1:1000; catalogue number: ab78364, Abcam, Cambridge, UK) for 24 h at room temperature and visualized with Alexa Fluor 594 donkey anti-mouse IgG secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. Subsequently, the sections were mounted, dried, and cover-slipped in antifade medium (Prolong Antifade Kit; Molecular Probes).
After identifying the preoptic sections with the most galanin-ir neurons in the ACN and the MPA in each of the five animals double labeled for galanin and oxytocin, we counted all galanin-ir neurons with an identifiable cell nucleus and all double-labeled cells. Counts were obtained using an Olympus BX60 light microscope with a 20× objective, fluorescent epi-illumination, and a filter that allows for simultaneous green and red visualization.