Preparation of digoxigenin (DIG)-labeled probes

Terminal transferase was used to label oligonucleotides at their 3′- ends by incorporation of a single DIG-labeled ddUTP (Roche Diagnostics, Mannheim, Germany). Briefly, 200 pmol oligonucleotides were incubated with 1 μL of 1 mM DIG-ddUTP and 20 U of terminal transferase (Roche) in 1 × reaction buffer and 5 mM CoCl₂ to a final volume of 20 μL at 37 °C for 60 min, and the reaction was stopped by adding 2 μL 0.2 M EDTA, pH 8.0. For the preparation of 18S rRNA probes, 18S rDNA was labeled by random priming with DIG-dUTP using DIG-High Prime kits (Roche). One microgram of 18S rDNA, amplified by polymerase chain reaction (PCR) and purified by agarose gel extraction, was mixed with the 5 × random primer mix supplied, and incubated at 37 °C overnight. The primers used for 18S rDNA amplification by PCR were 5′–AAC CTC GGG CCG ATC GCA CG–3′ and 5′–TCA AAG TAA ACG CTT CGG GC–3′.