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

Abstract

BackgroundOver the past five years in situ hybridization techniques employing tyramide amplification reagents have been developed and promise the potential detection of low/single-copy nucleic acid sequences. However the increased sensitivity that tyramide amplification brings about may also lead to problems of background staining that confound data interpretation.MethodsIn this study those factors enabling background-free biotinyl-tyramide based in situ hybridization assay of formalin-fixed paraffin-embedded tissues have been examined. SiHa, HeLa and CaSki cell lines known to contain HPV integrated into the cell genome, and archival cervical pre-invasive lesions and carcinomas have been successfully assessed using biotinylated HPV and centromeric probes.ResultsThe single most important factor both for sensitivity and clean background was a tissue unmasking regimen that included treatment with 10 mM sodium citrate pH 6.0 at 95°C followed by digestion with pepsin/0.2 M HCl. Concentrations both of probe and primary streptavidin-peroxidase conjugate and pH of hybridization mix and stringency washes were also critical for sensitivity. Certain probes were more associated with background staining than others. This problem was not related to probe purity or size. In these instances composition of hybridization mix solution was especially critical to avoid background. 3-amino-9-ethylcarbazole was preferred over 3,3'-diaminobenzidene as a chromogen because background was cleaner and the 1–2 copies of HPV16 integrated in SiHa cells were readily demonstrable. HPV detection on metaphase spreads prepared from SiHa cells was only successful when a fluorescent detection method was combined with tyramide reagent. 'Punctate' and 'diffuse' signal patterns were identified amongst tissues consistent with the former representing integration and 'diffuse' representing episomal HPV. Only punctate signals were detected amongst the cell lines and were common amongst high-grade pre-invasive lesions and carcinomas. However it remains to be determined why single/low-copy episomal HPV in basal/parabasal cells of low-grade lesions is not also detectable using tyramide-based techniques and whether every punctate signal represents integration.ConclusionsA tyramide-based in situ hybridization methodology has been established that enables sensitive, background-free assay of clinical specimens. As punctate signals characterize HPV in high-grade cervical lesions the method may have potential for clinical applications.

Detection of specific human papillomavirus types in paraffin‐embedded sections of cervical carcinomas

“…No PCR or HPV typing was performed. Evans et al [2003] performed studies to optimize tyramide-amplified in situ hybridization signal in paraffin-embedded tissue sections of cervical lesions and in cell lines. They identified assay parameters that permitted a low degree of background in these tissues.…”

In Situ Hybridization Signal Patterns in Recurrent Laryngeal Squamous Papillomas Indicate that HPV Integration Occurs at an Early Stage

“…HPV type was determined by dot blot hybridization with oligonucleotide probes [12,13] In Situ Hybridization (ISH) ISH was carried out on 34/35 FFPE sections (papilloma lesion from one specimen was exhausted during sectioning for DNA extraction). A tyramide-based ISH assay (GenPoint TM , Dako North America, Carpinteria, CA) was performed using a Dako biotinylated wide spectrum HPV probe and 3-amino-9-ethylcarbazole (AEC) as the substrate [15]. Cervical lesions were used as ISH positive controls, and ISH was performed minus HPV probe in the hybridization mix as a negative control.…”

“…Cervical lesions were used as ISH positive controls, and ISH was performed minus HPV probe in the hybridization mix as a negative control. The data was recorded in terms of diffuse signal patterns (consistent with episomal DNA), and punctate signals (consistent with HPV integrated into host cell chromosomes) [15,16]. Non-detection of HPV by ISH was considered consistent with a low-copy number HPV infection below the detection threshold sensitivity of the ISH technique.…”

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

“…Most preparations of paraffin melt at 581C therefore dewaxing methods using xylene washes of different lengths (5, 10, 20, 30 and 40 min) at both RT, 42 and 581C were analysed. Suboptimal proteolytic digestion (over or under digestion) with pepsin may lead to absence of signals, loss of morphological detail and/or reduced specificity of the probe (eg, multiple signals of varying sizes in normal cells, nonspecific spots juxtaposed to the nuclear envelope and increased background 23 ). Therefore, we tested varying digestion times between 4 and 10 min.…”

“…23 Factors known to affect the ability of the probe to hybridize specifically to a single genomic region and to reduce nonspecific background staining and nonspecific signals were tested, including the deparaffinization (dewaxing) method, digestion time, probe concentration, hybridization buffer and posthybridization washes. 23 The effect of the number of overlapping BACs on the signal size was also investigated.…”