3h microscale autoradiography standards

Brain regional D1DR-binding densities were examined as described previously [23 (link),26 (link)]. Slide-mounted sections were incubated in Tris-buffer (50 mM Tris–HCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, and 1 mM MgCl₂; pH 7.7) containing 0.2 nM [3H]SCH 23390 (specific activity 66.0 Ci/mmol, Amersham (Amersham, UK)) for 90 min at room temperature. Sections for non-specific binding were incubated in Tris-buffer containing 0.2 nM [3H]SCH 23390 and 10−7 M cis-flupenthixol. After incubation, the slides were drained, washed twice for 5 min in buffer at +4 °C and briefly dipped twice into distilled water (+4 °C). Sections were dried at room temperature overnight and exposed to a tritium-sensitive film (3H Hyperfilm^®, Amersham) at −20 °C for 6 weeks, together with Amersham 3H Microscale Autoradiography Standards^®.

Brain regional D2DR-binding densities were examined as described previously [23 (link),26 (link)]. Slide-mounted sections were incubated in Tris-buffer (50 mM Tris–HCl containing 120 mM NaCl; pH 7.4) containing 0.4nM [3H]spiperone (specific activity 109.0 Ci/mmol, Amersham) for 60 min at room temperature. Sections for non-specific binding were incubated in Tris-buffer containing 0.4 nM [3H]spiperone, 10−5 M haloperidol, and 10−5 M ketanserin. After incubation, the slides were drained, washed twice for 5 min in buffer at 4 °C and briefly dipped twice into distilled water (4 °C). Sections were dried at room temperature overnight and exposed to a tritium-sensitive film (3H Hyperfilm^®, Amersham) at −20 °C for 3 weeks, together with Amersham 3H Microscale Autoradiography Standards^®. After the development, all films were digitized, and the images were processed in ImageJ.