Fluorescent In Situ Hybridization in Sole Larvae

Chromosome preparations were carried out as described by Rodriguez et al. [2 (link)], using larvae (1–3 days after hatching) of S. senegalensis. The DNA-BAC was purified using Plasmid Midi Kit (Quiagen, Hilden, Germany) following manufacturer’s instructions, and then labelled using Biotin or Digoxigenin Nick Translation Mix (Roche Molecular Biochemical), as described by manufacturer’s instructions. Pre-treatment of chromosome preparations and hybridization were carried out following the protocol described by García-Cegarra et al. [30 (link)]. For the immunocytochemistry detection, the antibodies described in Rodriguez et al. [2 (link)] were used. The antibodies were prepared in Tween Non-Fat Milk (TNFM, 4x SSC, 0.05% Tween 20.5% skim milk). Chromosome staining was carried out with 0.4 mg/mL of 4’,6-diamidino-2-phenylindole DAPI-Vectashield (Antifade Mounting Medium) (Vector), and the images were examined with a Zeiss Palm MicroBeam microdissector and fluorescence microscopes equipped with an AxioCam MRm digital camera.