CEP: Key Product Information

CEP: 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.*vysis-products-cep-protocol-1.gif
  • Centrifuge for 1-3 seconds, vortex and then re-centrifuge.
  • Heat for 5 minutes in a 73°C water bath, and then place on a slide warmer set to 45-50°C.

*For simultaneous hybridization of two CEP probes, substitute 7 µL CEP hybridization buffer, 1 µL purified H2O, and 1 µL each of two different CEP DNA probes.

 

2. Slide Preparation

  • Mark hybridization areas with a diamond tipped scribe. vysis-products-cep-protocol-2.gif
  • Immerse the slide in the 73+/-1°C denaturant bath (70% formamide/2X SSC) for 5 minutes.
  • Dehydrate slide 1 minute in 70% EtOH, 1 minute in 85% EtOH, and 1 minute in 100% EtOH.
  • Dry slide and place on slide warmer (45-50°C) for up to 2 minutes.

 

3. Hybridization

  • Apply 10 µL of probe mix to slide. Apply coverslip immediately upon vysis-products-cep-protocol-3.gifplacing probe on slide. (Slide does not require sealing during short hybridizations).
  • Place slide in a pre-warmed non-humidified box and allow for hybridization to proceed for at least 30 minutes (but not more than 60 minutes) in a 42°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 the coverslip and immediately place into 0.4X SSC/0.3%NP-40 wash tank, agitating the slide for 1-3 seconds. Repeat for the other three slides, and then leave all four slides in the coplin jar for 2 minutes.
  • 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

vysis-products-cep-protocol-4.gif
  • 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 vysis-products-cep-protocol-5.gif

  • Store slide at -20°C in dark. 
 
 
 
 
CEP Procedure Notes

 

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 first opening each vial of reagent, centrifuge for 2-3 seconds using a standard bench-top microcentrifuge. 

 

Place room temperature Coplin jar containing the denaturant solution in a 73+/-1°C water bath approximately 30 minutes prior to use. 

 

If more than four slides have been hybridized they must be washed in more than one batch. The temperature of the wash solution must return to 73+/-1°C before washing each batch.

 

 
 
 
 
CEP Preparations of Working Reagents

 

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/0.37 NP-40 Wash Solution 

Add H2O to bring final volume of the solution to 1 liter. Filter through 0.45um pore filtration unit. Store up to six months at room temperature. Note: The wash solution must be at 73+/-1°C before using. In order to maintain the proper temperature range, four slides MUST be placed in the heated solution at one time. If fewer than four slides have been hybridized, room temperature microscope slides must be used to bring the number to four slides. If more than four slides have been hybridized they must be washed in more than one batch. The temperature of the wash solution must return to 73+/-1°C before washing each batch. 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.

 

Denaturing Solution (for 70 mL) 

Add 49 mL formamide, 7 mL 20X SSC (pH 5.3) and 14 mL purified H2Oto 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.

 
 
 
 

You are about to exit the Abbott family of websites for a 3rd party website

Links which take you out of Abbott worldwide websites are not under the control of Abbott, and Abbott is not responsible for the contents of any such site or any further links from such site. Abbott is providing these links to you only as a convenience, and the inclusion of any link does not imply endorsement of the linked site by Abbott. The website that you have requested also may not be optimised for your screen size.

Do you wish to continue and exit this website?

Yes No

You are about to enter an Abbott country or region specific website.

Please be aware that the website you have requested is intended for the residents of a particular country or countries, as noted on that site. As a result, the site may contain information on pharmaceuticals, medical devices and other products or uses of those products that are not approved in other countries or regions.

Do you wish to continue and enter this website?

Yes No