Fluorescent in situ Hybridization (FISH)
Reagents and Equipment
- 20x Saline-sodium citrate buffer (SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7, or Product No. S6639)
- RNase A (Product No. R4642) 100 Āµg/mL in 2x SSC
- Pepsin (Product No. P6887) 40 units/mL in 10 mM HCl
- Paraformaldehyde, EM grade (Product No. P6148) freshly depolymerized, 4% w/v in water
- Ethanol
- Labeled probe. Plasmid DNA is labeled with biotin-11-dUTP using nick translation random priming or the A3803 (e.g., ADVANCEā¢ Nick Translation Kit)
- Hybridization mix solution: 50% formamide (Product No. F7508), 10% dextran sulfate (Product No. D8906), 0.1% SDS (Product No. L4390), 0.5-1.5 ng/Āµl labeled probe and 300 ng/mL Salmon Sperm DNA (Product No. D7656) in 2x SSC
- Wash buffer: 20% formamide (Product No. F7508) in 0.1x SSC
- Detection buffer: 0.2% Tween 20 (Product No. P1379) in 4x SSC
- Blocking buffer: 5% bovine albumin (Product No. A3803) in detection buffer
- Antibody or detection compound (e.g., Streptavidin-Cy3, Product No. S6402) in blocking buffer
- DAPI (Product No. D9542) 2 Āµg/mL in antifade mounting medium.
- Fluorescence microscope, filters and optional triple band pass filter (x58, Omega Optics)
- Glass slides (Product No. S8400)
- Plastic cover slips for incubation and hybridization steps (cut from autoclavable waste bags, e.g., Product No. B4408)
- Heat block/ modified thermocycler
- Coplin jars for washing steps (Product No. S6016, S5641 or S5891)
Procedure
Slide Preparation
Hybridization
Detection
- Start with chromosome preparations from any cell type.
- Incubate with 200 ĀµL RNase for 1 hour at 37 Ā°C
- Wash slides in 2x SSC for 5 minutes. Repeat.
- Rinse slides in 10 mM HCl.
- Incubate with 200 ĀµL pepsin for 10 minutes at 37 Ā°C.
- Rinse slides in deionized H2O.
- Wash slides in 2x SSC for 5 minutes. Repeat.
- Stabilize slides in paraformaldehyde for 10 minutes.
- Wash slides in 2x SSC for 5 minutes. Repeat.
- Dehydrate slides in an ethanol series: 70%, 80%, 95%; 2 minutes each.
- Air dry.
- Prepare 30 Āµl hybridization solution per slide. Heat to 70 Ā°C. for 10 minutes and place on ice.
- Place 30 Āµl of hybridization solution on each slide and cover with a plastic cover slip.
- Denature slide at 65-70 Ā°C for 5 minutes on heat block.
- Gradually decrease temperature to 37 Ā°C.
- Hybridize at 37 Ā°C overnight in humidity chamber.
- Wash slides in 2x SSC to remove coverslip.
- Wash slides in wash buffer at 40 Ā°C for 5 minutes. Repeat.
- Wash slides in 0.1x SSC at 40 Ā°C for 5-15 minutes.
- Wash slides in 2x SSC at 40 Ā°C for 5-15 minutes.
- Cool slides to room temperature.
- Equilibrate slides in detection buffer for 5 minutes.
- Block in blocking buffer for 20-30 minutes.
- Incubate with 50 Āµl antibody or detection compound for 30-60 minutes (e.g., 5 Āµg/ml Streptavidin-Cy3 in blocking buffer).
- Wash slides in 2x SSC for 5 minutes. Repeat twice.
- Counterstain with DAPI solution for 10 minutes.
- Rinse briefly and mount in antifade mounting medium.
- Analyze with fluorescence microscope.
Materials
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References
1.
Heslop-Harrison J, Schwazarcher T, Anamthawat-JĆ³nsson K, Leitch AR, Shi M. 1991. In situ hybridization with automated chromosome denaturation. Technique. 3109-15.
2.
Leitch AR. 1994. In situ hybridization : a practical guide. Oxford (England): BIOS Scientific Publishers.
3.
Harris N, Oparka KJ. 1994. Plant Cell Biology: A Practical Approach. Oxford (England): Oxford University Press.
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