Fluoro image analyzer fla5100

Each brain section was incubated in 40% (v/v) DMSO solution containing
[¹²⁵I]ligand (10 kBq, 0.02 nM) for
1 h. The slices were rinsed for 5 min, two times each,
with 70% (v/v) DMSO solution, and subsequently dipped into cold water for
30 s. The sections were dried under a steam of cold air and placed
in contact with imaging plates (BAS-MS 2040; Fujifilm Corp., Tokyo, Japan) for
24 h. Distribution of radioactivity on the plates were analysed
using the Fluoro Image Analyzer (FLA5100; Fujifilm Corp.). Thereafter, serial
sections were also analysed by immunohistochemical staining of
PrP^Sc deposition as described above.

DNA (dried, 1μg) was dissolved in a succinate buffer (pH 6.0) containing 10 mM CaCl₂ and digested with 0.001 units of spleen phosphodiesterase II and 0.02 units of micrococcal nuclease in 4 μl total volume for 5 h at 37°C. DNA digest was labeled with 1μCi [γ-P³²]ATP (6000 Ci/mmol) and 1.5 units of T4 polynucleotide kinase in 10 mM bicine-NaOH pH 9.7 buffer containing 10 mM MgCl₂, 10 mM DTT, and 1 mM spermidine. After 0.5 h at 37°C, apyrase (10 units/ml) in the same buffer was added and incubated for another 0.5 h. The 3’nucleotide phosphate was cleaved off with 0.2 μg RNase P1 in 500 mM ammonium acetate buffer, pH 4.5. Identification of [γ-P³²]m⁵C was performed with two-dimensional thin-layer chromatography (TLC) on cellulose plates using the solvent system isobutyric acid:NH₄OH:H₂O (66:1:17 v/v) in the first dimension and 0.2 M sodium phosphate (pH 6.8)-ammonium sulfate-n-propyl alcohol (100 ml/60 g/2 ml) in the second dimension. Radioactivity was subsequently measured using a Fluoro Image Analyzer FLA-5100 with Multi Gauge 3.0 software (FujiFilm). Each analysis was repeated three times. For precise calculations, we evaluated spots corresponding not only to m⁵C, but also to products of its degradation, such as cytosine (C) and thymine (T). The amount of m⁵C was calculated as R = [(m⁵C/m⁵C+C+T)]*100 (23 (link)).