Amersham typhoon ip biomolecular imager

Formalin-fixed paraffin-processed sections were rehydrated and equilibrated in PBS for 30 minutes. Sections were then incubated with 100 kBq/mL of ¹⁸F-sodium fluoride in PBS for 1 hour at room temperature with a blocking control (10 µmol/L sodium fluoride) before two 5-minute washes in PBS and one in deionized water. Dried sections were exposed to a high-resolution autoradiography plate (BAS-IP-SR 2040; Cytiva), which were imaged on an autoradiography imager (Amersham Typhoon IP Biomolecular Imager, Cytiva).
Sample ¹⁸F-sodium fluoride content was quantified using the FIJI software (v 2.0.0, open source). Gray scale images were imported and a region of interest drawn around the perimeter of the image to provide mean background activity. Regions of interest were drawn around individual samples providing a mean gray intensity. The result was adjusted by dividing by the background activity, standardizing measurements, and allowing samples across separate experiments to be compared.

Radiolabeled RNA from in vitro transcription reactions was processed and analyzed exactly as described previously (43 (link)). Briefly, 150 μl samples (25 μl sample +125 μl Stop Solution) were mixed with an equal volume (150 μl) of Tris (pH 8) buffered phenol:chloroform:isoamyl alcohol (25:24:1, v/v), mixed by vortexing and inversion, and centrifuged at 18,500g and 4 °C for 5 min. The aqueous phase was collected and transferred to a new tube. Nucleic acids were precipitated by adding three sample volumes (450 μl) of 100% ethanol and 1 or 1.2 μl of GlycoBlue Coprecipitant and chilling at −70 °C for at least 30 min. The samples were centrifuged at 18,500g and 4 °C for 30 min, the supernatant was removed, the samples were centrifuged again briefly to pull down residual ethanol, and residual ethanol was removed. The pellets were dissolved in 6.5 μl of formamide loading dye, denatured by heating at 95 °C for 5 min, loaded on a prewarmed 7.5 M urea, 12% polyacrylamide, 35 × 43 cm, 0.4 mm thick sequencing gel in a Model S2 Sequencer Apparatus, and run at 1400 V for 2.5 to 3 h. The resulting gel was exposed to a storage phosphor screen for 12 to 16 h, and the storage phosphor screen was scanned using an Amersham Typhoon IP Biomolecular Imager (Cytiva).