Pretreating Paraffin Specimens

Specimen slides of paraffin sections have been prepared in advance and have been baked at 56° C. You will proceed through the remainder of the pretreatment protocol and then perform hybridizations with Her-2/neu / CEP® 17 probes.

Deparaffinizing Slides

Hemo-De is a non-toxic solvent with properties similar to xylene and is used to dissolve paraffin. Ethanol dehydrates the specimen and solubilizes the remaining paraffin, making it suitable for pretreatment.

1. Immerse Slides in Hemo-De for 10 minutes at ambient temperature.

2. Repeat twice using new Hemo-De each time.

3. Dehydrate slides in 100% EtOH for 5 minutes at ambient temperature.

4. Repeat Step 3.

5. Air dry slides or place slides on a 45-50° C slide warmer for 2-5 minutes. 

Pretrating Slides

HCL aids in solubilizing basic nuclear proteins, thus improving the accessibility of the DNA to the probes. HCl is also used to extract extracellular matrix proteins which limit accessibility of the probe to the cells and cause tissue autofluorescence. The pretreatment buffer aids in denaturing proteins and removing crosslinks between proteins. This is important to permit sufficient protease digestion.

1. Immerse slides in 0.2N HCl for 20 minutes.

2. Immerse slides in purified water for 3 minutes.

3. Immerse slides in Wash Buffer for 3 minutes.

4. Immerse slides in Pretreatment Solution at 80° C for 30 minutes.

5. Immerse slides in purified water for 1 minute.

6. Immerse slides in Wash Buffer for 5 minutes. Repeat using the second jar of Wash Buffer. 

Treating Slides with Protease

The protease serves to digest tissue proteins and is essential for accessibility of the probes to the target DNA.

1. Remove slides from the second jar of Wash Buffer and remove excess buffer by blotting the edges of the slides on a paper towel.

2. Immerse slides in Protease solution at 37° C for 10 minutes.

3. Immerse slides in Wash Buffer for 5 minutes. Repeat using the second jar of Wash Buffer.

4. Dry slides on a 45-50° C slide warmer for 2-5 minutes.

Fixing the Specimen

Specimen fixation ensures that tissue loss in denaturation and hybridization is minimal.

1. Immerse the slides in 10% buffered formalin at ambient temperature for 10 minutes.

2. Immerse the slides in Wash Buffer for 5 minutes. Repeat using the second jar of Wash Buffer.

3. Dry slides on a 45-50° C slide warmer for 2-5 minutes. Proceed with the Vysis LSI® probe protocol.

Denaturing the Specimen

1. Place slides into prewarmed Denaturation Solution at 72° ± 1°C(<6/jar) for 6 minutes.

2. Dehydrate in 70%, 85% and 100% EtOH, 1 minute each.

3. Dry on a 45-50°C slide warmer for 2-5 minutes.

Hybridization

1. Prewarm the probe to room temperature, vortex and spin down.

2. Add 10 ml of probe mix on the sample area of the slide.

3. Place 22x22 mm coverslip and seal edges with rubber cement.

4. Place slides in the prewarmed humidified chamber with a tight lid at 37°C incubator overnight (14-18 hours). 

Post-hybridization Washing

1. Add post-hybridization wash buffer to each of the two Coplin jars. Prewarm one jar in the 72±1°C waterbath and the second one at room temperature.

2. Remove the rubber cement seal from the slide by gently pulling up on the sealant with forceps.

3. Immerse slide(s) in post-hybridization wash buffer at room temperature and float off coverslip.

4. Remove excess liquid by wicking off the edge of the slide and immerse slide in post-hybridization wash buffer at 72±1° C for 2 minutes (£6 slides/jar).

5. Remove slide(s) from the wash buffer, air dry in the dark in an upright position.

Counterstain and SlideStorage

1. Apply 10 ml of DAPI counterstain to the target area of the slide and apply a glass coverslip. Store the slide(s) in the dark prior to signal enumeration. Or Store in next step.

2. Store hybridized slides (with coverslips on) at -20°C in the dark. After removing from -20°C storage, allow slide(s) to reach room temperature prior to viewing using fluorescence microscopy.

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