Fluorescence In Situ Hybridization for MicroRNA Detection in Archived Oral Cancer Tissues

Figure 1

Simplified scheme of fluorescence in situ hybridization (FISH) for microRNA detection. Locked nucleic acid (LNA)-incorporated oligonucleotides with Watson-Crick complimentary sequence against mature microRNA was used as probe, and the 5′ end of probe was labeled with digoxigenin (DIG). Hybridization was performed at 50°C. After successful binding of such probes to their target sequence, and stringent washing steps to remove excess and nonspecifically bound probes, sequentially added were mouse anti-DIG antibody, horseradish peroxidase (HRP)-conjugated anti-mouse IgG (or HRP-anti-digoxigenin antibody in place of the 2 antibodies), and the HRP substrate Cyanine 5-conjugated tyramide. Thereafter, the positive signal could be visualized by fluorescence microscopy with proper filter sets as described in Materials and Methods section.