These experiments were performed as described by Kitchen and colleagues (Kitchen et al., 1990; Slowe et al., 1999). Animals were killed by decapitation, and brains were removed and immediately immersed in isopentane in a stainless steel beaker on dry ice. Coronal sections (20 μm) were cut on a cryostat (Leica CM3050 S) maintained at −20 °C, thaw-mounted onto gelatin-subbed slides, and stored at −80 °C until processed. Sections were thawed, rinsed in 50 mM Tris–HCl buffer (pH 7.4), and incubated with 5 nM [3H]DAMGO in 50 mM Tris–HCl buffer for 1 h at 25 °C. Non-specific binding was assessed in the presence of 10 μM naloxone. Slides were then rinsed three times (2 min each) in cold 50 mM Tris–HCl buffer, pH 7.4, and once (30 s) in de-ionized H2O. Slides were dried under a cool stream of air and exposed to tritium-sensitive storage phosphor screens for 1 week in cassettes along with [3H]microscale for calculation of results.
