Skip to main content

Table 1 Summary of Optimized Protocol for FFPE Tissues

From: Optimization of biotinyl-tyramide-based in situ hybridization for sensitive background-free applications on formalin-fixed, paraffin-embedded tissue specimens

1. Cut sections onto ADCELL™ coated slides using molecular biology grade water.
2. Dewax and treat with 10 mM sodium citrate pH6.0 at 95°C.
3. Digest with 100μg/ml pepsin/0.2 M HCL at room temperature.
4. Treat slides with 0.6%H2O2/methanol.
5. Apply probe/hybridization mix (pH7.0) to section, seal under coverslip, heat denature at 95°C.
6. Hybridize overnight at 37°C.
7. Remove coverslip under 2X SSC/0.05% Tween20 pH7.0 at room temperature.
8. Wash slides in 0.2X SSC/0.05% Tween20 pH7.0 at 55°C.
9. Apply primary streptavidin-peroxidase conjugate (DAKO) diluted 1:500 (HPV) or 1:5000 (centromeric probes).
10. Apply biotinyl-tyramide and secondary streptavidin peroxidase (DAKO).
11. Demonstrate hybridization signal with 3-amino-9-ethylcarbazole (AEC) applied for ~10 minutes.