2.4. Fluorescence In Situ Hybridization

sGISH was performed following the protocol described by Falistocco [12]. The slides were pretreated with 100 μg/mL of RNase A in 2× SSC (0.3 M NaCl, 0.03 M sodium citrate) for 1 h at 37 °C and washed three times in 2× SSC. After incubation with 80 units/mL of pepsin (Sigma-Aldrich) in 10 mM HCl for 15 min at 37 °C, the chromosome preparations were stabilized by immersion in freshly depolymerized 4% (w/v) paraformaldehyde in water for 10 min at room temperature, washed in 2× SSC, dehydrated in a graded ethanol series, and air-dried. The hybridization solution was similar for FISH and GISH experiments with the exception of the concentration of probes and the salmon sperm DNA that was not used for GISH. It consisted of 2 ng/μL of ribosomal and telomeric probe or 5 ng/μL of the genomic probe, 50% (v/v) formamide, 10% (w/v) dextran sulphate, 0.1% (w/v) sodium dodecyl sulfate (SDS), and 300 ng/μL sheared salmon sperm DNA. The hybridization mixture was pre-denatured for 10 min at 70 °C and chilled on ice, then it was applied to the slides and denatured together with chromosome preparations at 70 °C in a modified thermocycler for 5 min. Then, the temperature was gradually decreased to 37 °C. The hybridization was carried out overnight at 37 °C. Post hybridization washes were carried out in 20% formamide (v/v) in 2× SSC at 42 °C. The same procedure as described for GISH was used for in situ hybridization of clone pTa71 and telomeric sequences labelled with digoxigenin and biotin, respectively. Detection of digoxigenin- and biotin-labelled probes was carried out with anti-digoxigenin conjugated with FITC (Roche) and streptavidin conjugated with Cy3 (Sigma-Aldrich), respectively.

All the preparations were counterstained with 2 μg/mL of 4′,6-diamidino-2- phenylindole (DAPI) and then mounted in antifade solution Vectashield (Vector Laboratories, Petergorough, UK).

Karyograms were obtained by arranging the chromosomes into eight homology groups and disposing the groups according to decreasing length, with the exception of the satellite chromosomes which were assembled as group 8 regardless of length.

The slides were examined with a Microphot Nikon epifluorescence microscope (Tokyo, Japan). Images were recorded with a SONY digital photocamera (Tokyo, Japan) and then processed using Adobe Photoshop CS3 (San Jose, California, USA).