“…RNA in situ hybridization for high-risk HPV E6/E7 mRNA was performed using the RNAscope HPV-HR18 Probe (Advanced Cell Diagnostics, Hayward, CA). Tumor sections were cut at five microns, pretreated with heat and protease, and hybridized with a single cocktail probe recognizing 18 high-risk HPV genotypes (16,18,26,31,33,35,39,45,51,52,53,56,58,59, 66, 68, 73, and 82). Preamplifier, amplifier and horseradish peroxidase-labeled probes were sequentially hybridized, and color was developed with diaminobenzidine.…”