Fluoroshield mounting medium with dapi stain

The slides were stained with 5% Giemsa solution. Selected metaphases were captured using a Zeiss Axio Imager Z2 (Zeiss, Oberkochen, Germany), equipped with a Metafer-MSearch automatic scanning platform (MetaSystems) and CoolCube 1 b/w digital camera (MetaSystems). Karyograms were prepared using the Ikaros karyotyping platform (MetaSystems). At least 10 metaphases per individual were studied. We performed C-banding stain to detect heterochromatin distribution according to the protocol of Sumner [46 (link)]. The slides were first treated with 0.2 N HCl for 30 min at room temperature, then with prewarmed 5% Ba(OH)₂ for 10 min at 45 °C and finally in 2× SSC for 1 h at 60 °C. Subsequently, the slides were washed with distilled water, air-dried and stained with Fluoroshield mounting medium with DAPI stain (Sigma-Aldrich).

Chromosome suspensions were dropped onto glass slides and incubated at 60 °C for either 1 h or overnight prior to all cytogenetic experiments. The chromosome spreads were stained with 5% Giemsa solution (Penta Chemicals, Prague, Czech Republic) for 15 min to prepare the karyograms. C-banding stain was used to detect heterochromatin distribution and the W chromosome in Uroplatus following the protocol of Sumner [77 (link)]. Briefly, the chromosome spreads were first treated with 0.2 N HCl for 15 min at room temperature, then treated with prewarmed 5% Ba(OH)₂ for 10 min at 45 °C and finally treated with 2 × SSC for 1 h at 60 °C. Subsequently, the chromosome spreads were washed with distilled water, air-dried and stained with Fluoroshield mounting medium with DAPI stain (Sigma-Aldrich, St. Louis, MO, USA).