[F-18]-AV-1451 was synthesized as previously described by us.19 (link) [F-18]-AV-1451 phosphor screen autoradiography was performed following a slightly modified version of a previously published protocol.1 In brief, 10μm-thick frozen brain sections were fixed in 100% methanol at room temperature for 20 minutes and then transferred to a bath containing high specific activity [F-18]-AV-1451 in 10mM PBS with a radioactivity concentration of approximately 20 μCi/ml. Adjacent brain slices were placed in a bath that was identical in all aspects except that unlabeled AV-1451 was added to yield 1 μM chemical concentration, a blocking condition sufficient to saturate essentially all available specific binding sites of tau.1 After incubation for 60 minutes, racks of slides were removed from the respective radioactive solutions and briefly incubated in a series of wash baths to remove unbound radiotracer. Wash solutions and incubation times were: 10 mM PBS for 1 minute, 70% ethanol/30% PBS for 2 minutes, 30% ethanol/70% PBS for 1 minute, and lastly 100% 10 mM PBS for 1 minute. Racks were removed from the final wash solution and slices were allowed to air dry before transfer of the slides to a storage phosphor screen (Multi Sensitive Phosphor Screen, PerkinElmer Life and Analytic Sciences, Shelton, CT) that had been photobleached immediately prior by exposure on a white light box for a minimum of 15 minutes. The slides and phosphor screen were enclosed in an aluminum film cassette and set in a dark area away from sources of radioactivity for the duration of the overnight exposure period. Under dim lighting conditions, the cassette was opened and the slides removed from the exposed screen, which was mounted to the carousel of the digital imaging system (Cyclone Plus Storage Phophor Scanner, PerkinElmer Life and Analytic Sciences). Scanning of screens was controlled by the manufacturer’s OptiQuant software package using the highest available resolution of 600 dpi (approximately 42 micron sampling interval). Digital images were saved in uncompressed form at full resolution and pixel depth. Images from adjacent brain slices incubated in the unblocked (high specific activity [F-18]-AV-1451 only) and blocking ([F-18]-AV-1451 plus 1 μM unlabeled AV-1451) conditions were compared to determine total and non-specific binding of [F-18]-AV-1451 in the tissue.