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

BACKGROUND:
In the past five years, in situ hybridization techniques utilizing tyramide signal amplification have emerged as promising tools for detecting low or single-copy nucleic acid sequences. However, the increased sensitivity can also introduce background staining, complicating data interpretation.
METHODS:
This study optimized a biotinyl-tyramide-based in situ hybridization protocol for formalin-fixed, paraffin-embedded (FFPE) tissues to minimize background staining. Cell lines with known integrated HPV genomes (SiHa, HeLa, and CaSki) and archival cervical lesions and carcinomas were analyzed using biotinylated HPV and centromeric probes.
RESULTS:
The most critical factor for achieving both high sensitivity and low background was a tissue pretreatment step involving heat-induced antigen retrieval with 10 mM sodium citrate (pH 6.0) at 95 °C, followed by pepsin digestion in 0.2 M HCl. Optimal probe and streptavidin-peroxidase concentrations, along with carefully controlled hybridization and wash conditions (including pH and stringency), were also essential. Background staining varied by probe, independent of probe size or purity, and was largely influenced by hybridization buffer composition.
Among chromogens tested, 3-amino-9-ethylcarbazole (AEC) provided cleaner backgrounds than 3,3′-diaminobenzidine (DAB), and allowed clear visualization of the 1–2 integrated copies of HPV16 in SiHa cells. Detection of HPV on metaphase spreads from SiHa cells was only successful using a combination of fluorescent detection and tyramide amplification.
Two distinct signal patterns were observed in tissue samples: punctate signals, consistent with HPV integration, and diffuse signals, likely representing episomal HPV. Cell lines showed only punctate signals, which were also frequently observed in high-grade lesions and carcinomas. The absence of detectable episomal HPV in basal/parabasal cells of low-grade lesions remains unexplained, and the correlation between punctate signals and integration requires further investigation.
CONCLUSIONS:
A tyramide-amplified in situ hybridization protocol has been optimized to enable sensitive and background-free detection of HPV in clinical FFPE specimens. The presence of punctate signals in high-grade cervical lesions suggests potential utility of this method in clinical diagnostics.