Vector dab substrate kit for peroxidase

Amygdala tissue of the rats in Experiment 1 was processed for FOS expression. Ninety min after the start of the potentiated feeding test, the rats were killed by exsanguination under deep isoflurane anesthesia, and perfused with 0.9 % saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were removed, post-fixed and cryoprotected overnight in 4% paraformaldehyde in 0.1 M PB containing 12% sucrose, and stored at −80°C. Brains were then sliced on a freezing microtome and 30-μm coronal sections through the amygdala were collected for FOS immunohistochemical staining, which followed a protocol similar to that used by Lee et al. (2005) (link). The primary antibody was rabbit FOS antibody (1:5000 dilution; Santa Cruz Biotechnology, no. sc-52). After the primary antibody incubation (48–72 hrs at 4 °C), sections were rinsed in PBS, incubated in goat antirabbit IgG-biotinylated secondary antibody (1:250 dilution; Vector Laboratories) for 1–1.5 hrs, rinsed in PBS and then incubated in avidin–biotin peroxidase conjugate (Vector laboratories) for 1–2 hrs. After several rinses in PBS, sections were reacted using a Vector DAB substrate kit for peroxidase (Vector Laboratories) to visualize FOS. Sections were mounted on slides, dehydrated in ascending concentrations of alcohol, and coverslipped with Permount (Fisher Scientific).