Daniel:Protocols/FISH: Difference between revisions

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  7. Allow the rubber cement to air-dry for 5 minutes at room temperature
  7. Allow the rubber cement to air-dry for 5 minutes at room temperature
  8. Denature for 2.5 minutes at 92C; keep humid
  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''
''Day 2''
9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C
  10. The next day, remove coverslip carefully and wash slides in pre-warmed jar containing 2X SSCT at 60C for 15 minutes
  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
  11. Incubate slides in 2X SSCT at room temperature for 10 minutes

Revision as of 21:01, 25 July 2013

Fluorescence in situ Hybridization (FISH)

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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 

Slide Preparation

Original Protocol from Beliveau et al: File:Slide preparation for interphase FISH.pdf

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: File:Interphase FISH protocol.pdf

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. Allow slides 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