Aida image analyzer

Proteins separated by electrophoresis were transferred onto PVDF (polyvinylidene difluoride) membrane (Immobilon FL 0.45 µm, Merck) by semi-dry electroblotting (0.8 mA/cm², 1 h) using Transblot SD apparatus (Bio-Rad). Immunodetection was performed as described in [15 (link)]. Primary and secondary antibodies used for immunodetection are listed in Table S1. Signal detection was performed using fluorescence scanner Odyssey (LI-COR Biosciences, Lincoln, NE, USA). Signals detected by scanner were quantified by software AIDA Image Analyzer (Raytest Isotopenmessgeräte GmbH, Germany).

Male Sprague Dawley rats (250–300 g) were injected with 20 MBq of [¹⁸F]2b intra-venous under isoflurane (4%) anesthesia. For blocking studies BP897 (1 mg/kg) was administerd 30 min prior to radioligand injection. Rats were sacrificed by decapitation 30 min post radioligand injection and brains were removed and frozen in cooled hexane at −70 °C. Coronal and horizontal brain sections (12 µm) were cut on a cryostat microtome (HM550, Microm) and thaw-mounted on covered glass slides (Histobond^®). The glass slides were placed on a phosphor screen (Fujitsu) over night. The screens were analyzed with a DUERR Medical HD-CR 35 Bio (Raytest) and evaluated using the software AIDA image analyzer (Raytest). Afterwards the sections were HE stained and ascribed to figures in the rat brain atlas [42 ]. Binding in the regions of interest were evaluated as ratio of the activity in the region of interest through background activity. Binding in the cerebral matter of the respective sections was defined as background activity.

Male Sprague Dawley rats (200–250 g) were sacrificed by decapitation under deep isoflurane anesthesia and brains were removed and frozen in cooled hexane at −70 °C. Coronal brain sections (20 µm) were cut on a cryostat microtome (HM550, Microm-Thermo Fisher Scientific, Walldorf, Germany) and thaw-mounted on covered glass slides (Histobond^®, Marienfeld, Lauda Königshofen, Germany). The thaw mounted sections were stored at −20 °C up to 14 days.
The cryostat sections were pre-incubated for 15 min in incubation buffer (50 mM Tris-HCl, 40 mM NaCl, pH 7.4) at room temperature. The sections were then placed in incubation buffer containing 5 MBq of the respective tracer. For displacement studies BP897 (50 nM or 1 µM) was added. The sections were incubated at room temperature for 45 min and afterwards washed in ice cold incubation buffer for 3 × 5 min. Subsequently, they were dipped in water and after drying placed on a phosphor imaging screen (Fujitsu, Berlin, Germany) over night. The screens were analyzed with a DUERR Medical HD-CR 35 Bio (Raytest, Straubenhardt, Germany) and evaluated using the software AIDA image analyzer (Raytest). For anatomical identification of brain regions, the sections were compared with figures in the rat brain atlas [42 ].

Genomic DNA was treated with RNase H2 and separated by alkaline gel electrophoresis, essentially as described (Benitez‐Guijarro et al., 2018 (link)). In brief, genomic DNA (250 ng) was treated with 1 pmol of purified recombinant human RNase H2 (Reijns et al., 2011 (link)) and 0.25 μg of DNase‐free RNase (Roche) for 1 hr at 37℃ in 100 μl reaction buffer (60 mM KCl, 50 mM Tris–HCl pH 8.0, 10 mM MgCl₂, 0.01%Triton X‐100). Nucleic acids were ethanol precipitated and separated by alkaline gel electrophoresis (0.7% agarose, 50 mM NaOH, 1 mM EDTA). After electrophoresis, the gel was neutralized in 0.7 M Tris–HCl pH 8.0, 1.5 M NaCl and stained with SYBR Gold (Invitrogen). Images were taken using the FLA‐5100 imaging system (Fujifilm), and densitometry plots generated using AIDA Image Analyzer (Raytest).