Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences.

Conditions for combination of DNA in situ hybridization, using biotinylated DNA probes, with immunohistochemistry were investigated on cryostat sections, cytological preparations, and paraffin sections. We found that cryostat sections and cytological preparations are suitable for in situ hybridization of target DNA after fixation in acetone, methanol, ethanol, or Carnoy without further proteinase pretreatment. Acetone is also very suitable for immunostaining of cell surface or cytoskeleton antigens. We therefore performed combined immunoenzyme and in situ hybridization staining using this fixative. The best results were obtained when immunoperoxidase staining with diaminobenzidine/H2O2 was followed directly by in situ hybridization. In addition to immunoperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) staining with naphthol ASBI phosphate and New Fuchsin as a substrate could be used. In most instances, detection of the biotinylated hybrid with a streptavidin-biotinylated polyalkaline phosphatase method using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as the substrate was preferable. The double stainings were studied on the following test models: (a) frozen tonsil sections: cell surface antigens (pan T) and ribosomal DNA; (b) frozen genital condyloma sections; cytokeratins and human papillomavirus type 6 + 11 (HPV-6/11) DNA; (c) CaSKi cells: cytokeratins and HPV-16 DNA; (d) infected fetal lung fibroblasts: vimentin and cytomegalovirus (CMV) DNA. An adapted procedure was followed on routinely formaldehye-fixed and paraffin-embedded condyloma tissue. Immunoperoxidase staining for papilloma virus capsid antigen could be combined with DNA in situ hybridization with HPV-6/11 DNA. In this model, however, the accessibility of the target DNA had to be improved by enzyme treatment after the immunostaining and before starting the in situ hybridization.