Fluorescent In Situ Hybridization Protocol

FISH experiments were performed with eight satDNA families, which were common to the four analyzed species. We utilized primers described by Silva et al. (2017) (link) and probes were labeled with digoxigenin-11dUTP in PCR reactions. FISH experiments were performed following the protocol established by Pinkel et al. (1986) (link), with some modifications (Utsunomia et al., 2017 (link)). The metaphasic plate was treated with RNase A (50 μg/ml), for 50 min, with subsequent chromosomal DNA denaturation in 70% formamide/2 × SSC for 2 min, at 70°C. After hybridization, the slides were washed in 0.2 × SSC/15% formamide for 5 min at 42°C, with subsequent washes in 4 × SSC/0.5% Tween-20, at room temperature. Probe detection was performed with anti-digoxigenin-rhodamine (Roche, Basiléia, Switzerland) and the chromosomes were counterstained with DAPI (4ʹ,6-diamino-2-phenylindole, Vector Laboratories, Burlingame, United States). The results were analyzed using an optical microscope (Olympus BX61). Images were captured using the DP Controller software (Olympus®, Hamburg, Germany).