In Situ Hybridization for 28S rRNA

The in situ hybridization protocol was adapted from Reineke et al.^{56 (link)} Cells were fixed with 2% formaldehyde in PBS for 1 min at room temperature and permeabilized with ice-cold methanol for 10 min at −20 °C. The following 5′-biotinylated DNA probe was used to detect 28S rRNA: 5′-cggcgctgccgtatcgttccgcctgggc gggattctgacttagaggcgttc-3′. The probe was hybridized in hybridization buffer (50% formamide, 2 × SSC, 10% dextran sulfate, 0.2% BSA, 5 mM DTT) at a final concentration of 1 μg/ml for 24 h at 43 °C in a humidified chamber and detected by subsequent incubation with Cy3 streptavidin (GE Healthcare Amersham, Milan, Italy) in 4 × SSC plus 0.1% Triton X-100 for 1 h at room temperature. Blocking and incubation with primary and secondary antibodies were next performed as described above.