1. Detection buffer: 4 X SSC containing 0.2% (v/v) Tween 20.
  2. 2X SSC: dilute from 20X stock
  3. (Optional) alcohol series: 70%, 90%, 96% ethanol in water


  1. Slides after photography and analysis of the first probing results
  2. Single stranded or denatured hybridization mixture containing new labeled probe or probe combinations from Protocol 8.2 or 8.3


  • Blot immersion oil off surface of coverslip and carefully wipe around coverslip with solvent to remove any traces of oil from the slide. Wipe underneath of slide.
  • Put slides in 37 °C for 5-10min to reduce the viscosity of the glycerol/antifade mountant. Remove coverslip by lifting slowly but steadily with the edge of a razor blade (Note 1).
  • Wash slides in a staining jar with detection buffer at room temperature for 5 min followed by 2 times 30-60 min (Note 2).
  • Incubate slides in 2x SSC for 2 times 5 min at room temperature. Optionally dehydrate slides in an alcohol series two times each and then air-dry



  1. Do not dip slides with immersion oil into solutions to remove coverslip; an oil film will from on the surface of the solution and adhere to the preparation when taking it out. It is not possible to wipe off oil from coverslips completely without the likelihood of moving the coverslip and damaging the preparation
  2. The ‘washes' remove the antifade solution, but are unlikely to remove detection reagents and are not stringent enough to remove labeled probe. The denaturation stage will remove the labeled probe from the target, and the label will then be diluted in the new hybridization solution. Nevertheless, residual signal from the first probing will almost always be visible (also see Note 3).
  3. Denaturation by dipping the slide into a hot formamide solution (see Protocol 8.4 alternatives) removes more of the first probe, which goes into the formamide solution when denatured. Then add the hybridization mixture, coverslip and allow to hybridize.
  4. This protocol is based on Heslop-Harrison et al. (1992)