Wheat Genetics Resource Center

CHROMOSOME PREPARATION FOR IN SITU HYBRIDIZATION (ISH)

  1. Germinate seeds on moist filter paper in petri dishes at room temperature.
  2. Collect root tips when they are about 1 to 2 cm long and pretreat with ice water for 24 h.
  3. Fix in Carnoy's I solution for 1 to 7 days. Better ISH results are obtained after short fixation times, but squashes are difficult if the fixation time is less than 2 days.
  4. Stain the root tips in 1 % acetocarmine for 10 min to 1 h.
  5. Make squash preparations in 1 % acetocarmine but without boiling and heating.
  6. Store the slides in a -70°C freezer. Slides can be stored for many years without adversly affecting N-banding and ISH results.