3.4.2. In Vitro Autoradiographic Analysis of Binding Sites of [18F]**1** in Mouse Kidney

The kidneys was isolated after cervical dislocation from one CD-1 mouse, frozen rapidly in isopentane at −25 ◦C for 5 min, and stored at −25 ◦C until the sectioning. Cryosections (transversal, 10 μm) were obtained using a microtome (MICROM HM560, Thermo Scientific Microm, Fisher Scientific GmbH, Schwerte, Germany), mounted on microscopy slides (SuperFrost, Thermo Scientific Menzel, Fisher Scientific GmbH, Schwerte, Germany), dried for ~2 h at room temperature, and stored at −25 ◦C until the autoradiography study. For the experiment, the slides were taken out from the freezer, the cryosections dried under a stream of cold air, and pre-incubated with PBS for 15 min at room temperature. The incubation solution was decanted, the slices dried again under a stream of cold air, and covered afterwards with the incubation solution ([18F]**1**, 198 kBq/mL PBS or 1.19 nM at the time of incubation, without (total binding) or with co-incubation with 10<sup>−</sup>5, 10−7, and 10−<sup>9</sup> M α-CHC-Na). Incubation at room temperature was terminated after 60 min, the slides were washed twice in 50 mM

TRIS-HCl, pH 7.4 at 4 ◦C, on ice for two minutes each followed by dipping in ice-cold demineralized water for 5 s and rapid drying under a stream of cold air. Afterwards, the slides were exposed to a phosphor imager plate (BAS-IP TR 2025, FujiFilm Corporation, Tokyo, Japan) along with standards obtained by pipetting and drying 1 μL of each concentration of a serial dilution of the radiotracer solution on to a microscopic slide.
