Daniel:Protocols/FISH
Jump to navigation
Jump to search
Fluorescence in situ Hybridization (FISH)
Buffers
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
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
Slide Preparation
Original Protocol from Beliveau et al: Beliveau Protocol
1. Rinse slides in 100% ethanol; allow slides to dry completely 2. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media 3. Add 100 uL of cell suspension per slide roughly in the center 4. Allow cells to adhere for 1-3 hours at the growth temp of the cell line 5. Rinse slides briefly in 1X PBS 6. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS 7. Rinse briefly in 1X PBS in a coplin jar 8. Incubate for 5 minutes in 2X SSCT 9. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide 10. Transfer to fresh container of 2X SSCT + 50% (v/v) formamide for storage at 4C 11. Slides are typically best used within 1-2 weeks of creation
Hybridization
Original Protocol from Beliveau et al: FISH Protocol (Beliveau)
Day 1
1. Allow stored slides to warm to room temperature 2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in a pre-warmed coplin jar 3. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes 4. Remove slides and allow to cool to room temperature 5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip 6. Invert slides on to cocktail-covered coverslips; seal with rubber cement 7. Allow the rubber cement to air-dry for 5 minutes at room temperature 8. Denature for 2.5 minutes at 92C; keep humid 9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C
Day 2
10. The next day, remove coverslip carefully and wash slides in pre-warmed jar containing 2X SSCT at 60C for 15 minutes 11. Incubate slides in 2X SSCT at room temperature for 10 minutes 12. Incubate slides in 0.2X SSC at room temperature for 10 minutes 13. Tap slides dry on papertowels; do not let the surface containing the cells touch the paper towels 14. Add 15 uL mounting media such as SlowFadeGold with DAPI to a 22x30 #1.5 coverslip 15. Invert slides onto coverslips with mounting media 16. Seal each slide using nail polish 17. Allow at least 30 minutes for nail polish to dry 18. Slides are quite stable for several months at 4C
35mm Dish Sample Preparation
Slide Preparation
1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI 2. Add 20 uL of cell suspension per dish to the cener; add 80 uL RPMI 3. Allow cells to adhere for 6 hours at 37 C 4. Rinse slides briefly in 1X PBS 5. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS 6. Rinse briefly in 1X PBS 7. Incubate for 5 minutes in 1 mL of 2X SSCT 8. Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide 9. Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm 10. Slides are typically best used within 1-2 weeks of creation