FISH Procedure for Paraffin-embedded Tissue Sections

Reagents

• Paraffin Pretreatment Reagent Kit:
  • Pretreatment Solution (1N sodium thiocyanate): store at room temperature
  • Pepsin (Activity 1:3000 to 1:3500): Lyophilized, store at -20°C
  • Pepsin Buffer (NaCl solution, pH 2.0): store at room temperature
  • Wash Buffer (2X SSC, pH 7.0): store at room temperature
• Xylene: store at room temperature
• Ethanol (100%): store at room temperature
• Neutral buffered formalin solution (4% formaldehyde in PBS)
• Concentrated (12N) HCl
• 1N NaOH
• Ultra-pure formamide: see manufacturer’s recommendation for detailed information.
• 20X SSC salt: store at room temperature, avoid humidity
• NP-40: store at room temperature
• DAPI counterstain: store at 4°C in the dark
• Purified water

Working Solution Preparation

20X SSC Solution: Dissolve 66 g of 20X SSC powder in 200 ml of purified water. Bring the total volume to 250 ml with purified water. Adjust pH to 5.3 with concentrated HCl. The solution can be stored at room temperature for up to 6 months.

Denature Solution (70% formamide/2X SSC, pH 7.0-8.0): Mix 49 ml of formamide with 7 ml of 20X SSC, and bring the total volume to 70 ml with purified water. Store at 2-8°C in tightly capped container. The solution can be used for up to one week. Check pH within 7.0-8.0 before each use.

Pepsin Solution: Verify pH of Pepsin Buffer to be 2.0. Pre-warm 50 ml of Pepsin Buffer at 37°C. Dissolve 25 mg of pepsin in the Pepsin Buffer. Make fresh solution before each use.

Ethanol Solutions: Prepare v/v dilutions of 70%, 85%, and 100% ethanol with 100% ethanol and purified water. Store at room temperature in tightly capped container. Dilutions can be used for up to one week unless evaporation occurs or the solution becomes diluted due to excess use.

Post-Hybridization Wash Buffer (2X SSC/0.3% NP-40): Mix 3 ml of NP-40 with 100 ml of 20X SSC. Bring the total volume to 1,000 ml with purified water. Adjust pH to 7.0-7.5 with 1N NaOH. Discard used solution each day, store unused solution at room temperature for up to 6 months.

FISH Procedure for Paraffin-embedded Tissue Sections

Slide Pretreatment

1. Immerse slides in Xylene at room temperature (RT) for 10 minutes. Repeat twice with new Xylene each time.
2. Dehydrate slides in 100% ethanol at RT for 5 minutes. Repeat once with new 100% ethanol.
3.  Air dry slides or place slides on a 45-50°C slide warmer.
4. Immerse slides in 0.2N HCl at RT for 20 minutes.
5. Immerse slides in purified water at RT for 3 minutes.
6. Immerse slides in Wash Buffer at RT for 3 minutes.
7. Immerse slides in pre-warmed Pretreatment Solution at 80°C for 30 minutes.
8. Immerse slides in purified water at RT for 1 minute.
9. Immerse slides in Wash Buffer at RT for 5 minutes. Repeat once with new Wash Buffer.
10. Immerse slides in pre-warmed Pepsin Solution at 37°C for 10-60 minutes.
11. Immerse slides in Wash Buffer at RT for 5 minutes. Repeat once with new Wash Buffer.
12. Dry slides on a 45-50°C slide warmer for 2-5 minutes.
13. Immerse slides in neutral buffered formalin at RT for 10 minutes.
14. Immerse slides in Wash Buffer at RT for 5 minutes. Repeat once with new Wash Buffer.
15. Dry slides on a 45-50°C slide warmer for 2-5 minutes.

Probe Preparation

1. Pre-warm the probe at RT for 20-30 minutes.
2. Briefly vortex and spin down the probe.

Slide Denaturation & Hybridization

1. Immerse slides in pre-warmed denature solution at 72°C for 5 minutes.
2. Move slides from the denature solution immediately into 70% ethanol at RT for 1 minute with agitation.
3. Immerse slides in 85% ethanol at RT for 1 minute with agitation, followed by 100% ethanol for 1 minute.
4. Dry slides on a 45-50°C slide warmer for 2-5 minutes.
5. Apply 10 μl of probe mixture on each hybridization area and cover with a 22 mm x 22 mm coverslip. Seal coverslip(s) with rubber cement.
6. Place slides in a pre-warmed humidified hybridization chamber and incubate slides at 37°C overnight (14-18 hours).

Post-Hybridization Wash

1. Mark each hybridization area on the back of slides with a diamond-tip pen.
2. Carefully remove rubber cement.
3. Immerse slides in Post-Hybridization Wash Buffer at RT to loosen the coverslips. Shake gently to remove the coverslips; do not pull the coverslips off.
4. Immerse slides in pre-warmed Post-Hybridization Wash Buffer at 72°C for 2 minutes.
5. Air dry slides in the dark.
6. Apply DAPI counterstain and cover slides with coverslips.
7. Examine slides under a fluorescence microscope with proper filter sets.