Fluorescence In Situ Hybridization of Mitotic Chromosomes

Mitotic chromosomes were prepared from root tip meristems synchronized using 2.5 mM hydroxyurea and 2.5 μM amiprophos-methyl as described previously^{56 (link),65} . Probes were labeled with Alexa Fluor 568 or Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA) using nick translation⁶⁶ . The oligo-probes were labeled with biotin or fluorescein at their 5′ ends during synthesis (Integrated DNA Technologies, Leuven, Belgium). FISH was performed according to Macas et al.^{67 (link)} with hybridization and washing temperatures adjusted to account for AT/GC content and hybridization stringency allowing for 10–20% mismatches. The slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA), and examined using a Zeiss AxioImager.Z2 microscope with an Axiocam 506 mono camera. Images were captured and processed using ZEN pro 2012 software (Carl Zeiss GmbH).