LSI: Key Product Information

1. Probe Preparation

• At room temperature mix 7 µL of CEP hybridization buffer, 1 µL CEP DNA probe, and 2 µL purified H2O.*
• Centrifuge for 1-3 seconds, vortex and then re-centrifuge.
• Heat for 5 minutes in a 73°C water bath.
• Place on a slide warmer set to 45-50°C.

2. Slide Preparation

• Mark hybridization areas with a diamond tipped scribe.
• Immerse the slide in the 73+/-1°C denaturant bath (70% formamide/2X SSC) for 5 minutes. If metaphase chromosome morphology is problematic, a denaturant temperature of 70-73°C may provide for better results.
• Dehydrate slide 1 minute in 70% EtOH, 1 minute in 85% EtOH, and 1 minute in 100% EtOH.
• Dry slide and place on a  45-50°C slide warmer for up to 2 minutes.

3. Hybridization

• Apply 10 µL of probe mix to slide. Apply coverslip immediately upon placing probe on slide, and seal coverslip with diluted rubber cement.
• Place slide in a pre-warmed humidified box and allow for hybridization to proceed overnight for 12-16 hrs in a 37°C incubator.
• Prepare one wash tank with 0.4X SSC/0.3%NP-40 and place into the 73+/-1°C water bath for at least 30 minutes. Discard after 1 day of use. Prepare a second tank of 2X SSC/0.1% NP-40 at room temperature.
• Remove rubber cement seal and the coverslip and immediately place into wash tank (0.4X SSC/0.3%NP-40), agitating the slide for 1-3 seconds. Repeat to a maximum of four slides, then leave all slides in the coplin jar for 2 minutes. Do not remove the coverslips from several slides before placing any of the slides in the wash bath. Begin timing the incubation when the last slide has been added to the wash bath.
• Wash slide in 2X SSC/0.1% NP-40 at room temperature for 5 seconds to 1 minute,   agitating for 1-3 seconds as the slide is placed in the bath.
• Allow slide to air dry in darkness.

4. Interpretation

• Apply 10 µL of DAPI II counterstain to the target area of slide and add coverslip.
• View slide using a suitable filter set.

5. Storage

• Store slide at -20°C in dark.

Fluorophores are readily photo-bleached by exposure to light. To limit this degradation, handle all solutions containing fluorophores in reduced light, including those steps involved in handling the hybridized slide. Perform all steps which do not require light for manipulation (incubation periods, washes, etc.) in darkness.

The use of a calibrated thermometer is highly recommended for measuring the temperature of solutions. 

Prior to opening each vial of reagent, centrifuging for 2-3 seconds using a standard bench-top microfuge is recommended.

Place room temperature Coplin jar containing the denaturant solution in a 73°C water bath approximately 30 minutes prior to use.

If more than four slides have been hybridized they must be washed in morethan one batch. The temperature of the wash solution must return to73+/-1°C before washing each batch.

20X SSC (for 500 mL of 20X SSC, pH 5.3) 

Add 132g 20X SSC to 400 mL H2O and mix thoroughly. Adjust pH at room temperature with a pH meter to 5.3 using concentrated HCl and adjust to final volume of 500 mL. Filter through a 0.45 micron pore filtration unit. Store up to six months at room temperature.

2X SSC/0.1% NP-40 (for 1 liter)

Add 100 mL 20X SSC (pH 5.3) to 850 mL purified H2O. Add 1.0 mL NP-40. Adjust pH to 7.0-7.5 with NaOH using a pH meter. Add H2O to bring final volume of the solution to 1 liter. Store up to six months at room temperature.  

0.4X SSC/0.3% NP-40 Wash Solution

Mix thoroughly 20 mL of 20X SSC with 950 mL purified H2O. Add 3 mL NP-40. Mix thoroughly until NP-40 is dissolved. Adjust pH to 7.0 - 7.5 with NaOH. Add purified H2O to bring final volume to 1 L. Store at ambient temperature. Discard stock solution after 6 months, or if solution appears cloudy or contaminated.

Store up to six months at room temperature.

Denaturing Solution (for 70 mL)

Add 49 mL formamide, 7 mL 20X SSC (pH 5.3) and 14 mL purified H2O to a glass Coplin jar and mix thoroughly. Measure pH at room temperature using pH meter to verify pH is between 7.0-8.0. Use each batch of denaturant for seven days and then discard. Between periods of use, store at 4 C.

Ethanol Wash Solutions 

For final concentrations of 70%, 85% and 100%. Prepare v/v dilutions of 100% ethanol with H2O. Use dilution for up to seven days and then discard. If solution evaporates or becomes diluted, replace with fresh solution. Between periods of use, store at room temperature.