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.
- Incubate specimen slides in a Coplin jar containing 2X SSC Solution at 73°C for 2 minutes.
- Incubate slides in a Coplin jar containing Protease/Pepsin solution for 10 minutes at 37°C.
- Wash slides in a Coplin jar containing PBS at room temperature for 5 minutes.
- Place slides into a Coplin jar containing formaldehyde solution for 5 minutes at room temperature.
- Remove slides from the formaldehyde solution and wash in a Coplin jar containing PBS for 5 minutes at room temperature.
- Dehydrate slides for 1 minute in 70% ethanol, followed by 1 minute in 85% ethanol, and 1 minute in 100% ethanol.
- Air-dry slides.
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|
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
- 12N HCI (for adjusting pH of wash solutions)
- 1 N Na OH (for adjusting pH of wash solutions)
- Coplin jars
- Calibrated thermometer
- Graduated cylinder (100 to 1000 ml)
- Magnetic stirrer
- 100% ethanol
- 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)
- 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)
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.