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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.
The ToTelVysion Multi-color DNA FISH Probe Mixture consists of a total of 62 DNA FISH probes.
Component | ToTelVysion Multi-color DNA Probe |
Quantity | 30 µL x 15 vials containing various probe mixtures |
Composition | Fluorophore-labeled TelVysion, CEP® and LSI® probes and blocking DNA mixed with Hybridization Buffer containing Dextran sulfate, formamide and SSC (pH 7.0) |
Storage | -20°C protected from light |
TOTELVYSION VIAL # | PROBE MIXTURE DESCRIPTION |
Mixture 1 | TelVysion 1p SpectrumGreen, TelVysion 1q SpectrumOrange, TelVysion Xp/Yp SpectrumOrange and SpectrumGreen, CEP X SpectrumAqua |
Mixture 2 | TelVysion 2p SpectrumGreen, TelVysion 2q SpectrumOrange, TelVysion Xq/Yq SpectrumOrange and SpectrumGreen, CEP X SpectrumAqua |
Mixture 3 |
TelVysion 3p SpectrumGreen, TelVysion 3q SpectrumOrange, TelVysion 22q SpectrumOrange and SpectrumGreen, LSI bcr (22q11) SpectrumAqua |
Mixture 4 | TelVysion 4p SpectrumGreen, TelVysion 4q SpectrumOrange, TelVysion 21q SpectrumOrange and SpectrumGreen, LSI AML (21q22) SpectrumAqua |
Mixture 5 | TelVysion 5p SpectrumGreen, TelVysion 5q SpectrumOrange |
Mixture 6 | TelVysion 6p SpectrumGreen, TelVysion 6q SpectrumOrange, TelVysion 13q SpectrumOrange and SpectrumGreen, LSI 13 (13q14) SpectrumAqua |
Mixture 7 | TelVysion 7p SpectrumGreen, TelVysion 7q SpectrumOrange, TelVysion 14q SpectrumOrange and SpectrumGreen, LSI TCR (14q11.2) SpectrumAqua |
Mixture 8 | TelVysion 8p SpectrumGreen, TelVysion 8q SpectrumOrange, TelVysion 17p SpectrumOrange and SpectrumGreen, CEP 17 SpectrumAqua |
Mixture 9 | TelVysion 9p SpectrumGreen, TelVysion 9q SpectrumOrange, TelVysion 17q SpectrumOrange and SpectrumGreen, CEP 17 SpectrumAqua |
Mixture 10 | TelVysion 10p SpectrumGreen, TelVysion 10q SpectrumOrange, TelVysion 15q SpectrumOrange and SpectrumGreen, LSI PML (15q22) SpectrumAqua |
Mixture 11 | TelVysion 11p SpectrumGreen, TelVysion 11q SpectrumOrange, TelVysion 18p SpectrumOrange and SpectrumGreen, CEP 18 SpectrumAqua |
Mixture 12 | TelVysion 12p SpectrumGreen, TelVysion 12q SpectrumOrange, TelVysion 18q SpectrumOrange and SpectrumGreen, CEP 18 SpectrumAqua |
Mixture 13 | TelVysion 16p SpectrumGreen, TelVysion 16q SpectrumOrange |
Mixture 14 | TelVysion 19p SpectrumGreen, TelVysion 19q SpectrumOrange, LSI 19p13 SpectrumAqua |
Mixture 15 | TelVysion 20p SpectrumGreen, TelVysion 20q SpectrumOrange |
Materials Required But Not Provided
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
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.
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.
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
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.
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