Fluorescent in situ Hybridization (FISH)

Reagents and Equipment

1. 20x Saline-sodium citrate buffer (SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7, or Product No. S6639)
2. RNase A (Product No. R4642) 100 µg/mL in 2x SSC
3. Pepsin (Product No. P6887) 40 units/mL in 10 mM HCl
4. Paraformaldehyde, EM grade (Product No. P6148) freshly depolymerized, 4% w/v in water
5. Ethanol
6. Labeled probe. Plasmid DNA is labeled with biotin-11-dUTP using nick translation random priming or the A3803 (e.g., ADVANCE™ Nick Translation Kit)
7. 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/µl labeled probe and 300 ng/mL Salmon Sperm DNA (Product No. D7656) in 2x SSC
8. Wash buffer: 20% formamide (Product No. F7508) in 0.1x SSC
9. Detection buffer: 0.2% Tween 20 (Product No. P1379) in 4x SSC
10. Blocking buffer: 5% bovine albumin (Product No. A3803) in detection buffer
11. Antibody or detection compound (e.g., Streptavidin-Cy3, Product No. S6402) in blocking buffer
12. DAPI (Product No. D9542) 2 µg/mL in antifade mounting medium.
13. Fluorescence microscope, filters and optional triple band pass filter (x58, Omega Optics)
14. Glass slides (Product No. S8400)
15. Plastic cover slips for incubation and hybridization steps (cut from autoclavable waste bags, e.g., Product No. B4408)
16. Heat block/ modified thermocycler
17. Coplin jars for washing steps (Product No. S6016, S5641 or S5891)

Procedure

Slide Preparation
Hybridization
Detection

Slide Preparation

1. Start with chromosome preparations from any cell type.
2. Incubate with 200 µL RNase for 1 hour at 37 °C
3. Wash slides in 2x SSC for 5 minutes. Repeat.
4. Rinse slides in 10 mM HCl.
5. Incubate with 200 µL pepsin for 10 minutes at 37 °C.
6. Rinse slides in deionized H₂O.
7. Wash slides in 2x SSC for 5 minutes. Repeat.
8. Stabilize slides in paraformaldehyde for 10 minutes.
9. Wash slides in 2x SSC for 5 minutes. Repeat.
10. Dehydrate slides in an ethanol series: 70%, 80%, 95%; 2 minutes each.
11. Air dry.

Hybridization

1. Prepare 30 µl hybridization solution per slide. Heat to 70 °C. for 10 minutes and place on ice.
2. Place 30 µl of hybridization solution on each slide and cover with a plastic cover slip.
3. Denature slide at 65-70 °C for 5 minutes on heat block.
4. Gradually decrease temperature to 37 °C.
5. Hybridize at 37 °C overnight in humidity chamber.

Detection

1. Wash slides in 2x SSC to remove coverslip.
2. Wash slides in wash buffer at 40 °C for 5 minutes. Repeat.
3. Wash slides in 0.1x SSC at 40 °C for 5-15 minutes.
4. Wash slides in 2x SSC at 40 °C for 5-15 minutes.
5. Cool slides to room temperature.
6. Equilibrate slides in detection buffer for 5 minutes.
7. Block in blocking buffer for 20-30 minutes.
8. Incubate with 50 µl antibody or detection compound for 30-60 minutes (e.g., 5 µg/ml Streptavidin-Cy3 in blocking buffer).
9. Wash slides in 2x SSC for 5 minutes. Repeat twice.
10. Counterstain with DAPI solution for 10 minutes.
11. Rinse briefly and mount in antifade mounting medium.
12. Analyze with fluorescence microscope.
    

References

1. Heslop-Harrison J, Schwazarcher T, Anamthawat-Jónsson 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.