Fla 7000 bio imaging analyzer

Tumour cryosections were exposed to a phosphor image plate (Fuji Imaging Plate BAS-SR 2025, Fuji Photo Film Co., Ltd., Tokyo, Japan) together with a set of calibrated standards26 (link) for 12 h. After each exposure, the imaging plate was scanned with a computerised imaging analysis system (FLA 7000 Bio-Imaging Analyzer; Fuji Photo Film Co.) and images were analysed by Multi Gauge V3.2 (Fuji Photo Film Co.). To evaluate radioactivity derived from the fraction of FMISO covalently bound to macromolecules, the serial sections were washed with ethanol and tris-buffered saline once and three times, respectively, and then immersed in ethanol for 3 min to remove extractable ¹⁸F-FMISO. After sections were dried, ARG images were acquired as described above.

The purified recombinant protein (3 μg) was incubated in kinase buffer (20 mM HEPES, pH 7.5, 125 mM NaCl, 10 mM MgCl₂, 5 mM MnCl₂) and 10 μM ATP containing 1 μCi [γ-³²P]ATP (3000 Ci/mmol, IZOTOP, Japan) for 30 min at 25°C. The reactions were stopped by the addition of 2 × SDS sample buffer. The phosphorylated proteins were electrophoresed on a 10% SDS-PAGE gel. The total protein content of the gel was visualized by CBB staining, and the phosphorylated proteins were detected using an FLA 7000 Bio-Imaging Analyzer (Fuji, Japan).