3h microscale

Mice were terminally anaesthetized with an overdose of sodium pentobarbital and transcardially perfused with 0.9% saline. Brains were harvested, frozen in isopentane at a temperature of −40°C and stored at −80°C. NBH (n = 7), ME7 (n = 8) and ME7 + JNJ-527 (n = 8) mouse brains were coronally cryosectioned at 20 μm and directly mounted onto glass slides. Slides were incubated at room temperature for 30 min in 100 mM Tris-HCl containing 1 nM [3H]PK11195 (specific activity 82.7 Ci per mmol; Perkin Elmer), washed twice for 6 min in 100 mM Tris-HCl, rinsed dipping into dH₂O and air dried. Non-specific binding was carried out in the presence of 20 µM PK11195 (Sigma-Aldrich) and 1 nM ³H-PK11195. The slides were exposed to tritium-sensitive film (Amersham Hyperfilm MP, GE Healthcare) in autoradiography cassettes together with a set of tritium standards ([3H]Microscale, American Radiolabeled Chemicals) for 6 weeks. Sections for specific and non-specific binding were processed together in a paired protocol. Films were developed using ECOMAX X-ray film processor (PROTEC). Quantitative analysis was performed using a MCID image analyser (Image Research), and brain structures were identified using the mouse brain atlas of Franklin and Paxinos (1997). All regions of interest (hippocampus, cortex and thalamus) were analysed by freehand drawing tools in three consecutive sections per brain.