Aida 2

Cryosections of brains obtained from female athymic nude mice (Rj:NMRI-Foxn1 nu/nu) (10–12 weeks old, 25–38 g), were obtained as described above. The same protocol as in Section 4.3 was used. The incubation step was performed with 0.1–0.2 MBq/mL (S)-(−)-[¹⁸F]fluspidine in buffer for 60 min at room temperature. Nonspecific binding was determined in the presence of 10 µM of SA4503 (Tocris, Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany) or 100 µM to 10 nM of (S)-(−)-fluspidine, respectively. Developed autoradiographs were analysed in a phosphor imager (HD-CR 35; Dürr NDT GmbH & Co. KG, Bietigheim-Bissingen, Germany). The quantification was performed by using 2D-densitometric analysis (AIDA 2.31 software; raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany). The B_max and the K_D values were estimated by a linear regression model (equation: one-site binding (hyperbola)) using GraphPad Prism, Version 4.1 (GraphPad Inc., La Jolla, CA, USA).

Cryosections of brains obtained from female CD-1 mice (10–12 weeks) were thawed, dried in a stream of cold air, and pre-incubated in 50 mM TRIS-HCl buffer (pH = 7.4, 100 mM NaCl, 5 mM MgCl₂, 1 mM EDTA) containing 1 µU/mL adenosine deaminase (ADA) for 15 min at ambient temperature. Afterwards, brain sections were incubated with 0.1–0.2 MBq/mL [¹⁸F]FESCH in buffer for 90 min at room temperature. Non-specific binding was determined in the presence of 1 µM of FESCH or ZM241385, respectively. Subsequently, the sections were washed twice for 5 min in ice-cold TRIS-HCl buffer, and dipped for 5 s in ice-cold deionized water. The sections were rapidly dried in a stream of cold air before being exposed overnight on an imaging plate (Fujifilm Corporation, Tokyo, Japan). Developed autoradiographs were analyzed in a phosphor imager (HD-CR 35, Duerr NDT GmbH, Bietigheim Bissingen, Germany). The quantification was performed by using 2D-densitometric analysis (AIDA 2.31 software, raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany). Further data analysis was performed with GraphPad Prism, Version 4.1 (GraphPad Inc., La Jolla, CA, USA).