Daniel:Protocols/FISH: Difference between revisions

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  '''Hybridization Cocktail''': 2X SSCT, 50% formamide, 10% (w/v) dextran sulfate, 10ug RNAase A, 10-20 pmole probe  
  '''Hybridization Cocktail''': 2X SSCT, 50% formamide, 10% (w/v) dextran sulfate, 10ug RNAase A, 10-20 pmole probe  


==Slide Preparation==
===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'': [[Media:Slide_preparation_for_interphase_FISH.pdf|Beliveau Protocol]]
''Original Protocol from Beliveau et al'': [[Media:Slide_preparation_for_interphase_FISH.pdf|Beliveau Protocol]]
Line 25: Line 33:
  11. Slides are typically best used within 1-2 weeks of creation
  11. Slides are typically best used within 1-2 weeks of creation


==Hybridization==
===Hybridization===


''Original Protocol from Beliveau et al'': [[Media:Interphase_FISH_protocol.pdf|FISH Protocol (Beliveau)]]
''Original Protocol from Beliveau et al'': [[Media:Interphase_FISH_protocol.pdf|FISH Protocol (Beliveau)]]
Line 51: Line 59:
  18. Slides are quite stable for several months at 4C
  18. Slides are quite stable for several months at 4C


===Preparing the Hybridization Buffer===
==35mm Dish Sample Preparation==
 
==Slide Preparation==


  1. Combine probes into 1.5 mL eppendorf tube and dry in a vacuum centrifuge
  1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI
  2. Resuspend DNA in 12.5 uL 100% formamide; mix at 37C for at least 10 minutes
  2. Add 20 uL of cell suspension per dish to the cener; add 80 uL RPMI
  3. Add 12.5 uL 4X SSCT, 20% w/v dextran sulfate, 10 ug RNAase A
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

Revision as of 17:45, 4 February 2014

Fluorescence in situ Hybridization (FISH)

Back to Main

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