Bas sr 2040 imaging plate

The normal Balb/c mouse (male, 10–12 weeks-old) frozen brain sections (10-μm thickness) were prepared using a cryostat microtome (CM1900, Leica, Germany) one day before and kept in −80℃ freezer. The sections were first thawed at room temperature for 20 min and rehydrated with ice-cold PBS buffer (pH 7.4) for 5 min. Then all sections were incubated with 4 nM [¹⁸F]9e with, or without, 10 μM olaparib at room temperature for 1 h to define the control and nonspecific binding groups. After the radioligand incubation, all of them were washed three times with ice-cold PBS for 3 min, dried up with a fan, and exposed to a BAS-SR 2040 imaging plate (20×40 cm, Fujifilm, Japan) for 2 hr. In the end the plate were scanned with a Typhoon 7000 phosphorimager (GE Healthcare) in a condition of 500 V and a resolution of 25 μm.

Brain tissue from WT mice (female, 69.3 5 ± 4.7 weeks) were sagittally sectioned using a cryostat at 20 micron (Leica CM1850), mounted onto Superfrost™ Plus slides (Thermo Fisher Scientific, Waltham, MA) and stored at −80 °C until further use. On the day of the experiment, sections were thawed for 25 minutes, rehydrated in ice-cold phosphate buffered saline (PBS, pH 7.4) for 5 minutes, then incubated for 1 h at room temperature with the following concentrations of [¹⁸F]SynVesT-1: 100 nM, 50 nM, 25 nM, 10 nM, 5 nM, 2.5 nM and 1.0 nM in PBS, and adjacent sections were incubated with the same concentration range of [¹⁸F]SynVesT-1 containing 1 mM levetiracetam (an SV2A specific ligand). Brain sections were washed in ice-cold PBS (3×5 min), briefly dipped in D.I. water and dried in a fume hood for 1 h. A calibration standard, with eight dilutions, was prepared in duplicate from a starting [¹⁸F]SynVesT-1 activity of 1.0 MBq mL⁻¹. Air-dried sections along with the calibration standard were exposed to a BAS SR 2040 imaging plate (20 × 40 cm, Fujifilm, Japan) for 18 h. The plate was scanned with a Typhoon Phosphor Trio imaging system. Image analysis and quantification was carried out using Fiji (ImageJ, NIH).