Specimen pretreatment prior to codenaturation.

The following pretreatment procedure is highly recommended prior to hybridization of ToTelVysion probes on metaphase slide preparations. The purpose of this procedure is to make the chromosomal DNA accessible for hybridization and to protect the morphology of the chromosomes from the codenaturation process.

1. Incubate specimen slides in a Coplin jar containing 2X SSC Solution at 73°C for 2 minutes. 
2. Incubate slides in a Coplin jar containing Protease/Pepsin solution for 10 minutes at 37°C. 
3. Wash slides in a Coplin jar containing PBS at room temperature for 5 minutes. 
4. Place slides into a Coplin jar containing formaldehyde solution for 5 minutes at room temperature. 
5. Remove slides from the formaldehyde solution and wash in a Coplin jar containing PBS for 5 minutes at room temperature. 
6. Dehydrate slides for 1 minute in 70% ethanol, followed by 1 minute in 85% ethanol, and 1 minute in 100% ethanol. 
7. Air-dry slides.

The ToTelVysion Multi-color DNA FISH Probe Mixture consists of a total of 62 DNA FISH probes.

Materials Required But Not Provided

• 12N HCI (for adjusting pH of wash solutions) 
• 1 N Na OH (for adjusting pH of wash solutions) 
• Coplin jars
• Calibrated thermometer 
• Forceps 
• Graduated cylinder (100 to 1000 ml)
• Magnetic stirrer 
• 100% ethanol 
• Microcentrifuge 
• Microliter pipette tips for 1 to 10 µl volumes 
• Microliter pipettor for 1 to 10 µl volumes 
• pH meter 
• Precleaned glass microscope slides 
• Purified water (distilled or deionized) 
• Timer 
• Vortex mixer 
• Water bath (37°C and 73°C)
• Fluorescence microscope 
• 37°C incubator 
• 20X Standard Sodium Citrate (SSC), 500 g (List No. 02J10-032) NP-40, 4000 µl (List No. 07J05-001; Quantity 2)
• Formamide, Ultra Pure grade 
• DAPI II Counterstain 
• Slide warmer (45 to 50°C) 
• Immersion oil for fluorescence microscope 
• Rubber cement 
• Coverslips (12 mm round)
• Diamond tip scribe
• Disposable syringe
• pH indicator strips
• 0.45 uM filtration unit
• Humidified chamber
• Phosphate Buffered Saline (PBS) 
• 10% neutral buffered formalin 
• 2M Magnesium Chloride (MgCl2) 
• Protease/Pepsin

Preparing the Specimen Target

In order to hybridize all 15 ToTelVysion mixtures, use a minimum of 3 specimen slides with 5 scribed target areas per slide for a total of 15 target areas.

Example of Slide #1 containing targets 1, 2, 3, 4 and 5. Repeat same type of organization of targets for slides 2 and 3, with 5 targets on each slide.

Preparing the Probe and Adding the Probe Mixture to the Specimen Slide

1. Each ToTelVysion probe mixture is pre-mixed and prepared in hybridization buffer. Remove the vials from -20°C (±5°C). Thaw at room temperature. 
2. Centrifuge each of the thawed vials of probe mixture for 1 to 3 seconds in a microcentrifuge. 
3. Vortex and then briefly centrifuge again. 
4. Remove 3 µL of probe from the vial using a mlcropipettor and place into a microcentrifuge tube. Prepare probe from each of the 15 vials in this same manner. 
5. Place each of the microcentrifuge tubes containing the 3 µL of probe In a 73 ± 1°C water bath for 5 minutes to denature the probe. 
6. Remove tubes from water bath. 
7. Place tubes on a 45 to 50°C slide warmer until ready to apply probe to target DNA.

Note: If slides are ready when probe is denatured, you can apply probe immediately to target DNA. 

Hybridizing the Probe to the Specimen Target

Note: Prepare a humidified box by placing a paper towel moistened with water on the side of an airtight container. 

1. Remove the slides from the 100% ethanol. 
2. Dry slides by touching the bottom edge of the slides to a blotter and wiping the underside of the slides dry with a paper towel. 
3. Place slides on a 45 to 50°C slide warmer for up to 2 minutes to evaporate remaining ethanol. 
4. Apply 3 µL of probe mixture to 1 target area and immediately apply 12 mm round coverslip. Repeat for additional 14 target areas.

Note: Do not allow coverslips of each target area to touch. Capillary action between touching coverslips will cause probe from 1 target to move to the other.

5. Seal each coverslip with rubber cement, ensuring that the rubber cement overlaps the edge of the coverslip to create a tight seal between the coverslip and the slide surface. 

6. Place slides in the prewarmed humidified box and place box in a 37 °C incubator for 12 to 24 hours. 

Washing the Slide

Prepare the wash solutions:

Note: Bring solutions to appropriate temperature before beginning wash procedure.

• Pour 70 ml of 0.4X SSC/0.3% NP-40 into a Coplin jar. Place jar in a 73 ± 1°c water bath at least 30 minutes prior to use. Use 1 day, then discard.
• Pour 70 ml of 2X SSC/0.1 % NP-40 into a Coplin jar. Use at room temperature. Use 1 day, then discard.

Note: To maintain the proper temperature in 0.4X SSC/0.3% NP-40, wash 4 slides simultaneously. If you have less than 4 slides, add blank slides that are at room temperature to bring the total to 4.

 7.  Remove coverslips from a slide and immediately immerse slides in 0.4X SSC/0.3% NP-40 at 73 ±1°C. Agitate slides for 1 to 3 seconds. Repeat with other slides.  

8.   Remove slides after 2 minutes.

9.  Immerse slides in 2X SSC/0.1 % NP-40 at room temperature. Agitate slides for 1 to 3 seconds. Remove slides after 30 seconds to 2 minutes 

Visualizing the Hybridization

1. Air-dry slide in darkness.
2. Apply 3 µl of DAPI II counterstain to each target area of the slides and apply a 12 mm round coverslip. Alternatively, apply 10 µL of DAPI II counterstain to each half of the slides and apply a 22 x 50mm coverslip over the counterstain. Apply gentle pressure to the top of the coverslip to remove excess DAPI. Blot with paper towel. 

Setting the ThermoBrite Parameters

Immediately place each of the slides (with the probe and coverslip applied) onto the surface plate of the ThermoBrite. Moisten the humidity strips with water and place in the lid.

1. Seal each coverslip with rubber cement ensuring that the rubber cement overlaps the edge of the coverslip to create a tight seal between the coverslip and the slide surface.
2. For cultured lymphocytes and amniocytes, set the Denat Temp to 70 °C and the Denat Time to 3 minutes. (Note: These temperatures may need adjustment depending upon specimen type. Refer to the ThermoBrite Operator's Manual (55-005322).)
3. Set the Hyb Temp to 37 °C and the Hyb Time overnight (12 to 16 hours).
4. When the hybridization time is completed, wash the slides using the Rapid Wash Procedure as previously described.
5. Air dry slide in darkness.
6. Apply 3 µL DAPI II counterstain to each target area of the slide and apply a 12 mm round coverslip.