Table 1 Summary of Optimized Protocol for FFPE Tissues

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.