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The sensitivity of radiolabeled and digoxigenin-labeled RNA probes and synthetic oligonucleotide probes for the detection of seminal vesicle secretion protein II (SVS II) and androgen receptor (AR) mRNA was compared by in situ hybridization in paraformaldehyde-fixed cryostat sections of the rat prostate. Both genes are expressed in different amounts in the various prostatic lobes and contiguous glands. SVS II or AR RNA probes were either labeled with digoxigenin-11-UTP or [35S]UTP by in vitro transcription. A synthetic SVS II oligonucleotide probe was 3' end-labeled (tailed) with either digoxigenin-11-dUTP or [35S]dATP. Hybridized 35S-labeled probes were detected by autoradiography and digoxigenin-labeled probes by immunohistochemistry using alkaline phosphatase conjugated anti-digoxigenin antibody or gold-labeled antibody followed by protein A-gold and silver enhancement. Digoxigenin-labeled probes provided the same degree of sensitivity as their 35S-labeled counterparts for the detection by in situ hybridization of weakly and strongly expressed mRNA. Using both labeling methods, the SVS II RNA probes were more sensitive than the oligonucleotide probes and background labelling of the 35S-labeled oligonucleotide probe was high. The digoxigenin method produced less background with all probe types, hybridization signals showed higher resolution and results were obtained faster than with radiolabeled probes. The immunogold silver enhancement system provided the fastest detection of digoxigenin-labeled probes with a sensitivity and resolution similar to that provided by alkaline phosphatase anti-digoxigenin immunohistochemistry. It is concluded that digoxigenin probe labeling and detection provides a sensitive, reliable, and efficient alternative to radiolabeled probes for in situ hybridization of mRNA.

Different approaches to the in-situ polymerase chain reaction (in-situ PCR) were compared in the detection and in-situ localization of chromosomal translocations (t14; 18) immunoglobulin gene rearrangements and viral DNA (cytomegalovirus, hepatitis B-virus) in cell suspensions, cytospins and tissue sections. Single and multiple primer pairs were compared in the amplification step of indirect in-situ PCR and long genomic probes or internal oligonucleotide probes in the subsequent in-situ hybridization (ISH). For direct in-situ PCR, in which amplification products were directly labeled with digoxigen-in-11-dUTP during PCR and detected immunohistochemically, only single primer pairs were used for amplification. In-situ PCR yielded best results in the cell suspensions and worked less efficiently in cytospins or tissue sections. Quantification of the results obtained in artificial cell mixtures yielded only an approximate correlation between the number of expected and observed positive cells. The specificity of the results was greater with indirect in-situ PCR than direct in-situ PCR, where false positive results were frequent. Successful indirect in-situ PCR in tissue sections required the use of multiple primer pairs for amplification and genomic probes for detection by ISH. False positive results in direct in-situ PCR were caused by primer-independent, but DNA polymeraseand cycling-dependent incorporation of digoxigenin-labeled nucleotides into cellular DNA, possibly related to DNA repair and/or internal priming. Non-specific results were most marked in tissue sections and were much less frequent in cell suspensions. In-situ PCR includes a number of different techniques, which are not equally applicable to different starting materials. Accurate interpretation of the results requires vigorous controls.