Chromosome Preparation and FISH Labeling

The preparations of mitotic metaphase and meiotic pachytene chromosomes were conducted using modified Carnoy’s solution II in accordance with the work of Setiawan et al. [13 (link)]. The Cmcent probe was labeled with Biotin-Nick translation mix (Roche), whereas CmSat162 and CmSat189 were labeled with Dig-Nick translation mix (Roche). The FISH protocol as described by Setiawan et al. [13 (link)] was followed. For the pachytene chromosomes, the hybridization mixtures were added on the chromosome preparations, covered with a 22 x 40-mm cover slip and sealed with rubber cement. The slides were denatured on a hot plate at 80°C for 2–3 min. Finally, the slides were placed in a humidity chamber and incubated at 37°C overnight. Detection solutions of 126 μL [1% BSA in 4x SSC 125 μl + 0.4 μl/ml anti-digoxigenin rhodamine (Roche) 0.5 μL + 0.5 μg/mL biotinylated streptavidin-FITC (Vector Laboratories) 0.5 μl] were used and washed in 2x and 0.1x SSC for 3 min after incubation at 37°C for 30 min. Finally, the slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) in a VectaShield antifade solution (Vector Laboratories).