Dual labeling of Nociceptin (green) and enkephalin (red). 7 μm paraffin sections of human brain tissue were cut and floated onto positively charged slides. After being dewaxed and microwaved in 10mM citric acid (pH 6.0) for 20 minutes at 600W, sections were incubated with the rabbit polyclonal antisera overnight at 4°C, primary antibody was detected using biotinylated goat anti-rabbit IgG followed by incubation with peroxidase-conjugated avidin.

Binding of the primary antibody was visualized using 3-amino-9-ethylcarbazole (AEC) in acetate buffer. Afterwards, sections were rinsed, counterstained  with Mayer`s hematoxylin and mounted in Vectamount mounting medium.