Daniel:Protocols/FISH
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Fluorescence in situ Hybridization (FISH)[edit]
Buffers[edit]
2X SSCT: 0.3M NaCl, 0.03M NaCitrate, 0.1% Tween-20 0.2X SSC: 0.03M NaCl, 0.003M NaCitrate Hybridization Cocktail: 2X SSCT, 50% formamide, 10% (w/v) dextran sulfate, 10ug RNAase A, 10-20 pmole probe
Preparing the Hybridization Buffer[edit]
1. Combine probes into 1.5 mL eppendorf tube and dry in a vacuum centrifuge 2. Resuspend DNA in 12.5 uL 100% formamide; mix at 37C for at least 10 minutes 3. Add 12.5 uL 4X SSCT, 20% w/v dextran sulfate, 10 ug RNAase A
Glass Slide Sample Preparation[edit]
Slide Preparation[edit]
Original Protocol from Beliveau et al: Beliveau Protocol
- Rinse slides in 100% ethanol; allow slides to dry completely
- Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media
- Add 100 uL of cell suspension per slide roughly in the center
- Allow cells to adhere for 1-3 hours at the growth temp of the cell line
- Rinse slides briefly in 1X PBS
- Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
- Rinse briefly in 1X PBS in a coplin jar
- Incubate for 5 minutes in 2X SSCT
- Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide
- Transfer to fresh container of 2X SSCT + 50% (v/v) formamide for storage at 4C
- Slides are typically best used within 1-2 weeks of creation
Hybridization[edit]
Original Protocol from Beliveau et al: FISH Protocol (Beliveau)
Day 1
- Allow stored slides to warm to room temperature
- Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in a pre-warmed coplin jar
- Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes
- Remove slides and allow to cool to room temperature
- Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip
- Invert slides on to cocktail-covered coverslips; seal with rubber cement
- Allow the rubber cement to air-dry for 5 minutes at room temperature
- Denature for 2.5 minutes at 92C; keep humid
- Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C
Day 2
- The next day, remove coverslip carefully and wash slides in pre-warmed jar containing 2X SSCT at 60C for 15 minutes
- Incubate slides in 2X SSCT at room temperature for 10 minutes
- Incubate slides in 0.2X SSC at room temperature for 10 minutes
- Tap slides dry on papertowels; do not let the surface containing the cells touch the paper towels
- Add 15 uL mounting media such as SlowFadeGold with DAPI to a 22x30 #1.5 coverslip
- Invert slides onto coverslips with mounting media
- Seal each slide using nail polish
- Allow at least 30 minutes for nail polish to dry
- Slides are quite stable for several months at 4C
35mm Dish Sample Preparation[edit]
Slide Preparation[edit]
- Prepare a cell suspension of 500 000-2 000 000 cells/mL in culture media
- Add 20 uL of cell suspension per dish to the cener; add 80 uL culture media
- Allow cells to adhere for 6 hours at 37 C
- Rinse slides briefly in 1X PBS
- Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
- Rinse briefly in 1X PBS
- Incubate for 5 minutes in 1 mL of 2X SSCT
- Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide
- Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm
- Slides are typically best used within 1-2 weeks of creation
Hybridization[edit]
Day 1
- Allow stored samples to warm to room temperature
- Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
- Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
- Remove samples and allow to cool to room temperature
- Add 25 uL hybridization cocktail to center
- Add 22x22 #1.5 coverslip; seal with rubber cement
- Allow the rubber cement to air-dry for 5 minutes at room temperature
- Denature for 2.5 minutes at 92C; keep humid
- Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C
Day 2
- The next day, remove coverslip carefully and wash in pre-warmed 2X SSCT at 60C for 15 minutes
- Incubate in 2X SSCT at room temperature for 10 minutes
- Incubate in 0.2X SSC at room temperature for 10 minutes
- Dry with kimwipes; do not let the surface containing the cells touch the kimwipes
- Add 15 uL DRAQ5 (diluted 1:1000) to center of dish
- Cover with 22x22 #1.5 coverslip; seal with nail polish
- Allow at least 30 minutes for nail polish to dry
- Slides are quite stable for several months at 4C
Cell Preparations[edit]
10 mL Dish[edit]
- Detach cells with 0.5mL trypsin
- Resuspend with 9.5 mL media
- Recommend 50/50 cell culture/media ratio for dish prep