Adopted from Wu lab protocol. 01/23/13
- Briefly wash cells with 1x PBS (not PBT)
- Fix cells with 4% formaldehyde for 15 min
- Quick wash with 1x PBS
- Wash cells with 2x SSCT for 5 min
- Wash cells with 50% formamide/2x SSCT for 5min. Store at 4C if not needed right away.
- Remove coplin jar(s) containing slides from 4 °C allow them to warm to room temperature
- Incubate slides in 2X SSCT + 50% (v/v) formamide for 2.5’ at 92°C in a prewarmed coplin jar. (Heat jars in waterbath).
- Transfer slides to a coplin jar containing 2X SSCT + 50% formamide at 60°C and incubate for 20’.
- Remove the slides from the 60°C coplin jar and allow them to cool to room temperature
- 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
- Invert slides onto the cocktail-containing coverslips such that the area containing the cells is covered. Seal with rubber cement
- Allow the rubber cement to air-dry for 5’ at room temperature
- 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]
- Transfer slides to a humidified chamber and hybridize overnight at 37°C or 42°C (depending on probe length)
- The next day, remove the coverslip carefully and wash slides in a pre-warmed coplin jar containing 2X SSCT at 60°C for 15’
- Transfer slides to a coplin jar containing 2X SSCT at room temperature and incubate for 10’
- Transfer slides to a coplin jar containing 0.2X SSC at room temperature and incubate for 10’
- 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
- Add 15 μl of mounting media such as SlowFade Gold + DAPI to a 22×30 #1.5 coverslip
- Invert slides onto mounting media-containing coverslips such that the area containing the cells is centered beneath the coverslip
- Seal each slide using nail polish
- Allow at least 30’ for the nail polish to dry before imaging the slides to ensure no nail polish gets on the microscope objectives
- 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