Adjacent frozen Sects. (10 µm) of rat and pig pancreas as well as rat brain and excised INS1 xenografts (insulinoma/beta cell model) were prepared on cryostat (Cryostar NX70, Thermo fisher scientific) at − 20 °C and mounted on adhesion glass slides (Superfrost Plus, Menzel-Gläser, Germany) to be stored at − 20 °C. For in vitro autoradiography (ARG), the frozen slides were first preincubated in phosphate buffered saline (PBS 150 ml, pH 7.4) at room temperature for 10 min and washed with MQ water for 1 min. Next, [11C]UCB-J in PBS and ethanol (1 MBq/ml) was added, and the sections were incubated for 40 min. To remove the excess buffer and unbound [11C]UCB-J, the slides were washed three times in PBS and once in MQ water and dried in a laboratory drying oven (Termaks, Bergen, Norway) for 10 min. A set of calibration standards were created for quantification by pipetting 10 μl drops of the same stock onto absorbent chromatography paper. The slides were then exposed to a freshly erased storage phosphor screen (BAS-MS, Fujifilm) for two half-lives of 11C (40 min) and then scanned on a phosphor imager (Amersham Typhoon FLA 9500 Phospor Imager, GE) with 4000 sensitivity and 25 µm pixel size. The images were analyzed in ImageJ (National Institutes of Health, US) software.
Amersham typhoon fla 9500 phospor imager
Manufactured by GE Healthcare
The Amersham Typhoon FLA 9500 is a phosphor imager used for the detection and quantification of radiolabeled samples. It utilizes a laser-based scanning system to detect and digitize phosphor-labeled samples. The imager can analyze a variety of sample types, including gels, membranes, and microplates.
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2 protocols using amersham typhoon fla 9500 phospor imager
1
In Vitro Autoradiography of [11C]UCB-J in Tissue Sections
2
In vitro Radiotracer Binding Assay
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In vitro ARG was performed on the cell pellets and fibrotic human liver. Prior to starting the experiments, the sections were thawed to room temperature. They were then pre-incubated in 150mL of a solution of PBS and 1% bovine serum albumin (BSA) at room temperature for 15 min. Optionally, to study the specificity of the radiotracer 2μM of unlabeled Cys-Z09591 was added to the previous solution (blocking condition). After 15 min, [18F]AlF-RESCA-Z09591 was added to the solution at a concentration of 5nM (approximately 0,1 MBq/mL), followed by incubation for 60 min. To remove the unbound radiotracer, the sections were washed twice in cold PBS/BSA 1%, once in PBS and dipped in MQ water. The sections were then dried at 9 37°C for 10min. A set of calibration standards was created by adding 10uL of the incubation solution containing the radiotracer to absorbent paper. Both the slides and standards were exposed to a phosphor-imaging plate (BAS-MS, Fuji-film) overnight and scanned using a Phosphor Imager (Amersham Typhoon FLA 9500 Phospor Imager, GE). The resulting images were analyzed using ImageJ software (National Institutes of Health, US). Paraffin-embedded biopsies from the same fibrotic liver were also obtained and prepared for correlative histology.
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