FISH Pretreatment

Short version protocol should not replace the package insert.

Intended Use
To prepare cells fixed onto slides for use in fluorescence in situ hybridization (FISH) with Vysis^® probes.
Reagents Provided
Materials Required but not Provided

absolute ethanol (EtOH)

10% buffered formalin solution

Carnoy's fixative (Methanol: Glacial Acetic Acid (3:1))

purified water (distilled or deionized)

Coplin jars

15 mL conical centrifuge tubes

Polypropylene microcentrifuge tubes (1.5 mL)

37 °C water bath

Pretreatment Procedure
1. Allow slide(s) to completely dry at room temperature.

2. Immerse slide(s) in 2X SSC for 2 minutes at 73 ±1 °C

3.Immerse slide(s) in protease solution for 10 minutes at 37 °C. (Ensurethat the temperature of the buffer is 37 °C prior to adding 25 mg (onetube) protease.

4. Wash slide(s) in 1X PBS for 5 minutes at room temperature.

5.Fix slides in 1% formaldehyde for 5 minutes at room temperature. (Mixtogether 12.5 mL of 10% neutral buffered formalin, 37 mL of 1X PBS, and0.5 mL of 100X MgCl₂ (one tube).

6.Wash slides in 1X PBS for 5 minutes at room temperature.

7.Dehydrates slide(s) by immersing in 70% ethanol solution at roomtemperature. Allow the slide(s) to stand in the ethanol wash for 1minute. Repeat with 85% ethanol, followed by 100% ethanol.

8. Proceed with the appropriate Vysis probe protocol.

Short version protocol should not replace the package insert.

Intended Use
To prepare cells fixed onto slides for use in fluorescence in situ hybridization (FISH) with Vysis probes.
Reagents Provided
Materials Required but not Provided

absolute ethanol (EtOH)

10% buffered formalin solution

Carnoy's fixative (Methanol: Glacial Acetic Acid (3:1))

purified water (distilled or deionized)

Coplin jars

15 mL conical centrifuge tubes

Polypropylene microcentrifuge tubes (1.5 mL)

37 °C water bath