Dig nucleic acid detection kit

ISH for human ALU genomic repeats was performed as described elsewhere26 (link). Briefly, sections were deparaffinized and rehydrated. Matrix digestion was obtained by enzymatic treatment with 10 μg ml⁻¹ proteinase K in 0.1 M Tris-HCl, 50 mM EDTA, pH 8, for 30 min at 37 °C. Sections were immediately post fixed in 3% formaldehyde for 10 min, washed in PBS, dehydrated in an ethanol series and dried for 20 min at room temperature. After rehydration and equilibration in PBS, sections were acetylated in 0.25% acetic acid containing 0.1 M triethanolamine (pH 8) for 10 min, and pre-hybridized with 50% deionized formamide containing 4 × SSC at 37 °C for 15 min. Sections were covered with hybridization buffer (1 × Denhardt's solution, 0.2 mg ml⁻¹ denatured sheared salmon sperm DNA, 4 × SSC and 50% deionized formamide) containing 1 ng μl⁻¹ digoxigenin-labelled double-stranded DNA probe specific for human ALU genomic repeats and covered with a glass coverslip. Denaturation of both the probe and the genomic DNA template was achieved by heating the slides at 95 °C for 45 s. Hybridization was performed at 42 °C overnight. Sections were washed twice for 30 min at room temperature with 2 × SSC and 0.1 × SSC and for 30 min at 50 °C in 0.1 × SSC. Digoxigenin was detected using a commercially available kit (DIG Nucleic Acid Detection Kit, Sigma) according to the manufacturer's protocol.