Daniel:Protocols/FISH

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Fluorescence in situ Hybridization (FISH)[edit]

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In-Depth Protocol (Beliveau)

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

  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[edit]

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

  1. The next day, remove coverslip carefully and wash slides in pre-warmed jar containing 2X SSCT at 60C for 15 minutes
  2. Incubate slides in 2X SSCT at room temperature for 10 minutes
  3. Incubate slides in 0.2X SSC at room temperature for 10 minutes
  4. Tap slides dry on papertowels; do not let the surface containing the cells touch the paper towels
  5. Add 15 uL mounting media such as SlowFadeGold with DAPI to a 22x30 #1.5 coverslip
  6. Invert slides onto coverslips with mounting media
  7. Seal each slide using nail polish
  8. Allow at least 30 minutes for nail polish to dry
  9. Slides are quite stable for several months at 4C

35mm Dish Sample Preparation[edit]

Slide Preparation[edit]

  1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in culture media
  2. Add 20 uL of cell suspension per dish to the cener; add 80 uL culture media
  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

Hybridization[edit]

Day 1

  1. Allow stored samples to warm to room temperature
  2. Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
  3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
  4. Remove samples and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to center
  6. Add 22x22 #1.5 coverslip; 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

  1. The next day, remove coverslip carefully and wash in pre-warmed 2X SSCT at 60C for 15 minutes
  2. Incubate in 2X SSCT at room temperature for 10 minutes
  3. Incubate in 0.2X SSC at room temperature for 10 minutes
  4. Dry with kimwipes; do not let the surface containing the cells touch the kimwipes
  5. Add 15 uL DRAQ5 (diluted 1:1000) to center of dish
  6. Cover with 22x22 #1.5 coverslip; seal with nail polish
    1. Allow at least 30 minutes for nail polish to dry
    2. Slides are quite stable for several months at 4C

Cell Preparations[edit]

10 mL Dish[edit]

  1. Detach cells with 0.5mL trypsin
  2. Resuspend with 9.5 mL media
  3. Recommend 50/50 cell culture/media ratio for dish prep