Biotinylated donkey anti rabbit antibody

Coronal brain sections (30 μm) were collected in 1:12 series using a freezing microtome and stored at -20°C in cryoprotectant (0.1 phosphate buffer, 30% sucrose, 1% polyvinylpyrrolidone, and 30% ethylene glycol). For Fos immunoreactivity, a one in 12 series of brain sections were rinsed (5 min X 5) in 50 mM potassium phosphate-buffered saline (KPBS) (pH 7.2), followed by incubation in 1% hydrogen peroxide solution for 10 min to quench endogenous peroxides and subsequently rinsed in KPBS. Tissue was then incubated in blocking buffer (KPBS) plus 0.25% bovine serum albumin and 0.4% Triton X-100) for 1 h at room temperature followed by incubation with a c-Fos-specific antibody (1:2500; Santa Cruz Biotechnology, Santa Cruz, CA, no. sc-52) in blocking buffer overnight at 4° C. The next day, sections were rinsed in KPBS (5 min X 5 times) followed by 1 h incubation in biotinylated donkey anti-rabbit antibody (1:500; Vector Laboratories, Burlingame, CA; no. BA1000). Sections were washed in KPBS and reacted with avidin-biotin solution (1:1000; Vector Laboratories, Burlingame) for 1 h at room temperature. Sections were washed once more and c-Fos immunoreactivity was visualized with 0.02% 3, 3′-diaminobenzidine (DAB) (Sigma)/hydrogen peroxide in KPBS solution.