DNA FISH on coverglass

Adopted from Wu lab protocol.  01/23/13

  1. Briefly wash cells with 1x PBS (not PBT)
  2. Fix cells with 4% formaldehyde for 15 min
  3. Quick wash with 1x PBS
  4. Wash cells with 2x SSCT for 5 min
  5. Wash cells with 50% formamide/2x SSCT for 5min.  Store at 4C if not needed right away.
  6. Remove coplin jar(s) containing slides from 4 °C allow them to warm to room temperature
  7. Incubate slides in 2X SSCT + 50% (v/v) formamide for 2.5’ at 92°C in a prewarmed coplin jar.  (Heat jars in waterbath).
  8. Transfer slides to a coplin jar containing 2X SSCT + 50% formamide at 60°C and incubate for 20’.
  9. Remove the slides from the 60°C coplin jar and allow them to cool to room temperature
  10. Add 25 μl of a hybridization cocktail composed of 2X SSCT, 50% formamide, 10% (w/v) dextran sulfate, 10 μg RNase A, and 10-20 pmole of Oligopaints probe to a 22×22 #1.5 coverslip
  11. Invert slides onto the cocktail-containing coverslips such that the area containing the cells is covered. Seal with rubber cement
  12. Allow the rubber cement to air-dry for 5’ at room temperature
  13. Denature for 2.5’ at 92°C by placing slides top of a water-immersed heat block inside a closed water bath (trying to keep temperature as uniform as possible via humidity).
  • [Work around: Place slide face up on top of inverted 92C heat block with a little bit of water to seal the slide to the heat block for 2.5 min]
  1. Transfer slides to a humidified chamber and hybridize overnight at 37°C or 42°C (depending on probe length)
  2. The next day, remove the coverslip carefully and wash slides in a pre-warmed coplin jar containing 2X SSCT at 60°C for 15’
  3. Transfer slides to a coplin jar containing 2X SSCT at room temperature and incubate for 10’
  4. Transfer slides to a coplin jar containing 0.2X SSC at room temperature and incubate for 10’
  5.  Remove slides from the coplin jar and gently tap them dry on paper towels – take care to just tap the thin edge of the slide and to never allow the surface containing the cells to directly contact the paper towels
  6. Add 15 μl of mounting media such as SlowFade Gold + DAPI to a 22×30 #1.5 coverslip
  7. Invert slides onto mounting media-containing coverslips such that the area containing the cells is centered beneath the coverslip
  8. Seal each slide using nail polish
  9. Allow at least 30’ for the nail polish to dry before imaging the slides to ensure no nail polish gets on the microscope objectives
  10. In our hands, slides remain quite stable (e.g. for several months) when stored at 4°C and protected from light

2X SSCT – 0.3 M NaCl, 0.03 M NaCitrate, 0.1% Tween-20 0.2X SSC – 0.03 M NaCl, 0.003 M NaCitrate

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