Alu positive control probe 2

In situ hybridization was carried out on Ventana discovery XT™ using the DABMap Detection System according to manufacturer's recommendations. Five micrometer thick sections were deparaffinized. Antigen retrieval was performed with RiboCC™ (pH 6.0) for 60 min at 95°C followed by digestion with Protease 3 (760–2020, Ventana) for 20 min at 37°C. Alu Positive Control Probe II (780–2221, Ventana) was dropped, slides were warmed at 85°C to denature DNA, hybridization was performed at 47°C for 1 h. Slides were washed twice with sodium saline citrate. Nonspecific binding was impeded by incubation of inhibitor D and endogenous biotins were blocked with avidin–biotin kit (760–050, Ventana). Bound probe was detected by rabbit anti-digoxigenin (780–4335, Ventana) and then tissues were incubated with biotinylated anti-rabbit Ab (Vector Laboratories). Slices were exposed at complex avidin–horseradish peroxidase at room temperature and finally were incubated with diaminobenzidine and hydrogen peroxide.