Chromosome preparation for in situ hybridization (ISH)
- Germinate seeds on moist filter paper in petri dishes at room temperature.
- Collect root tips when they are about 1 to 2 cm long and pretreat with ice water for 24 h.
- Fix in Carnoy's I 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.
- Stain the root tips in 1 % acetocarmine for 10 min to 1 h.
- Make squash preparations in 1 % acetocarmine, but without boiling and heating.
- Store the slides in a -70 C freezer. Slides can be stored for many years without adversly affecting N-banding and ISH results.
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Kansas State University | Wheat Genetics Resource Center
31 January, 1997.