Simultaneous application ofin situ DNA hybridization and immunohistochemistry on one tissue section

Abstract

Combined application of a non-radioactive in situ DNA hybridization procedure and the immunoperoxidase technique on one tissue section is described. Of six potential protocols, only one proved to be successful. First, the immunohistochemical procedure including visualization of enzyme activity is performed; the in situ DNA hybridization protocol is then applied. Using this protocol, several antigens, detected with monoclonal antibodies, and target DNAs, detected by using biotinylated human cytomegalovirus or human papilloma virus type 16 DNA probes, could be distinguished by their peroxidase activity (brown precipitate) and alkaline phosphatase activity (purple-blue precipitate) respectively. The method allows immunophenotyping of virus-infected cells as well as simultaneous visualization of two viral parameters. This technique has important implications for research and diagnostic purposes.

Colocalization of Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus 2 in Porcine Dermatitis and Nephropathy Syndrome by Double-Labeling Technique

“…Double-labeling techniques are used for the simultaneous demonstration of either two different cellular components or two different cell types or tissue structures. 20,24 The combination of immunohistochemistry and in situ hybridization allows the detection of nucleic acid and the simultaneous localization of antigen. The objective of the present study was to develop a double-labeling technique, combining immunohistochemistry and in situ hybridization techniques, to simultaneously demonstrate the cellular distribution of PRRSV antigen and PCV DNA in formalin-fixed, paraffin-embedded tissues from pigs with PDNS.…”

“…The initial application of immunohistochemistry avoids the possibility that the viral antigen could be denatured during the various incubation steps of the in situ hybridization procedure. 20,24 However, when peroxidase immunohistochemistry with diaminobenzidine (DAB) is performed, the DAB polymer may interfere with in situ hybridization. 3 Several investigators have demonstrated the superiority of digoxigenin-labeled probes over other nonradioactive detection systems for in situ hybridization.…”

A one-day double-labelling technique for tissue specimens: Immunogold-silver staining forin situ hybridization combined with alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for antigens

“…Due to the sensitivity of this method, various advances to increase the signal-to-noise ratio for the ISH as well as for a secondary double labelling step have been established. Amongst them are: 1. immunofluorescence or immunoperoxidase histochemistry followed by ISH using radioactive-labelled riboprobes with the option for quantification (Wolfson et al, 1985;Koenig et al, 1986;Shivers et al, 1986;Bursztajn et al, 1990); 2. the application of immunohistochemistry after ISH to reduce the problem of RNase interference (Chan-Paly et al, 1988;Hankin & Lloyd, 1989;Peltonen et al, 1989;Bugnon et al, 1991;Ehrlein et al, 1994;Heppelmann et al, 1994) and to avoid non-specific binding to diaminobenzidine precipitates (Soilberg et al, 1991); and 3. non-radioactive labelling ISH techniques (Wolber & Lloyd, 1988;Ironside et al, 1989;Van der Loos et al, 1989;Biffo et al, 1992;Morey et al, 1995).…”

Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors

“…Enzyme precipitates that withstand the proteolytic digestion and denaturation steps used in the ISH procedure, include the APase-fast red (Zheng et al 1993;Speel et al 1994a,c;Herbergs et al 1996a,b), APase-new fuchsin (Mullink et al 1989b;Porter et al 1990), APase-BCIP/NBT (Graham et al 1991), HRP-DAB (Mullink et al 1989b;Van der Loos et al 1989;Strehl and Ambros 1993), and β-Gal-BCIG ( Van den Brink et al 1990;Robben et al 1994;Speel et al 1994b) precipitates. In these procedures, the pretreatment and denaturation steps needed for optimal ISH after ICC remove the antibody and enzyme detection layers from their targets, but the precipitate remains firmly localized.…”

“…Images C, F-J are reproduced with permission of the Journal of Histochemistry and Cytochemistry, and images B, D, E are courtesy of J. Herbergs] less accessible to ISH reagents and to shield the target DNA ( Van der Loos et al 1989;Strehl and Ambros 1993), so that fine tuning of the ICC staining reaction is required to ensure proper ISH results.…”