Streptavidin biotin peroxidase kit

Coronal sections (14 μm) were cut in a microtome at −17°C and collected on Superfrost-plus slides (Thermo Fisher, Bartlesville, OK, USA). After Hemalun–Phloxine (HP) staining, anatomical areas (Fig. 2) were identified using the Paxinos and Watson rat brain atlas (Paxinos and Watson, 1998 ). To avoid counting the same neuron twice, three to six non-consecutive coronal sections of each brain region of interest (ROI) were processed for c-Fos immunohistochemistry. Sections were thawed and rinsed in 0.1 M PBS for 5 min and placed in a 10 mM citrate bath for 20 min at 100°C. Endogenous peroxidase activity was inhibited by adding 3% H₂O₂ in methanol for 15 min. Sections were rinsed in 0.1 M PBS for 15 min and incubated with a rabbit polyclonal anti-c-Fos antibody (1:500, Santa Cruz, Dallas, TX, USA) overnight at 4°C and processed with the Dako streptavidin-biotin-peroxidase kit according to the manufacturer’s instructions (Dako, Carpinteria, CA, USA). Binding was revealed using 3,30-diaminobenzidine (DAB). Negative controls were obtained by omitting the primary or secondary antibodies during the immunohistochemical procedure.