1. Single stranded or denatured hybridization mixture from Protocol
  2. Fixed and pretreated spread chromosome preparations from Protocol


  • Add appropriate hybridization mixture to each marked area on preparations. Cover with a small plastic coverslip. Make sure no bubbles are trapped underneath the coverslip. They can be easily freed by lifting the coverslip carefully (Note 1).
  • Heat slides to denature target on a heated block at 60-90 ° C, leave them for 5-10 min and then cool them down to 37 ° C (slow cooling is usually used) (Notes 2 and 3)
  • Thermal cyclers have the most accurate temperature control, but a heated plate with good temperature control can be used. Alternatively, a water bath at 90 °C with a lid (essential to maintain a high temperature) can be used. Slides are rested on glass rods lying on moist paper in a closed heat resistant chamber floating in the water bath, with a digital thermometer probe inside the chamber next to the slides. The temperature of the chamber is equilibrated and the temperature within the chamber accurately controlled by opening and closing the lid of the water bath for denaturation
  • Incubate slides at 37 °C (42 °C may be used to increase stringency) in a humid chamber, in the thermal cycler or on the heated block, typically for 16-20h (Note 4).
  • Check slides do not dry out or accumulate condensation.
  • Proceed to stringent washing (Protocol 9.1).



  1. For hybridization times up to 20 h, sealing of coverslips is not necessary. However, for hybridization times of several days, use silconized glass coverslips and seal them with rubber cement to avoid drying out.
  2. The range of time and temperature has to be adjusted to different species and material: 75°C for 5 min provides conditions that denature most DNA
  3. During denaturation, it is important that no condensation drips onto the slides or the hybridization mixture will be diluted and stringency decreased
  4. Hybridization time depends on the amount and length of probe and amount of target. For highly repetitive sequences, 2 - 4 h will be adequate, but it can be advantageous to leave single-copy sequences to hybridize for 72 h or more.
  5. The protocol described here is based on Heslop-Harrison et al. (1991).