A highly sensitive nonisotopic detection method for in situ hybridization

In situ hybridization is a technique that allows detection of specific DNA and RNA sequences in tissue sections. Nonisotopic techniques are fast and g ive a precise localization of the hybridization product, but a drawback is the low sensitivity. However, the sensitivity is dependent on the detection system used. To evaluate a sensitive in situ hybridization method with non radioactive probes we compared three different detection systems, using bio tin-labeled human papillomavirus (HPV) 16 probes. The three detection syste ms included (i) STAV-FITC method (streptavidin-fluorescein isothiocyanate/a lkaline phosphatase anti-FITC), (ii) APAAP method (mouse anti-biotin/anti-m ouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase), and (iii) t yramide signal amplification (TSA) method (STAV-horseradish peroxidase (HRP )/biotinyl tyramide/ STAV-HRP). The in sim hybridization methods were teste d on CaSki and SiHa cells and two cervical carcinomas known to be HPV16 pos itive. The cells and tissues had been fixed in 4% buffered formalin and par affin embedded. The three different detection systems gave satisfactory nuc lear staining in CaSki cells (CaSki cells contain >500 copies of HPV16 DNA) and the two cervical carcinomas. However, demonstration of HPV16 DNA in Si Ha cells (SiHa cells contain one to two HPV16 genome copies) was possible o nly by use of the APAAP method. it was concluded that the APAAP method prov ides the best sensitivity among the nonisotopic detection systems and can d etect single viral copies in formalin-fixed and paraffin-embedded material.