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

Reagent Quantity Composition Storage
Pepsin Buffer 3 x 50 mL 10 mM HCl 2 to 25 °C
Protease 3 x 25 mg 2500 - 3000 U/mg protease, lyophilized -20 to 8 °C
PBS 2 x 250 mL  1X PBS 2 to 25 °C
100X MgCl2 3 x 0.5 mL 2M MgCl2 * 6 H2O 2 to 25 °C
20X SSC 1 x 66g Sodium Chloride & Sodium Citrate -25 to 30 °C

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
  • Prolypropylene 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. (Ensure that the temperature of the buffer is 37 °C prior to adding 25 mg (one tube) 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. (Mix together 12.5 mL of 10% neutral buffered formalin, 37 mL of 1X PBS, and 0.5 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 room temperature. Allow the slide(s) to stand in the ethanol wash for 1 minute. Repeat with 85% ethanol, followed by 100% ethanol.
  8. Proceed with the appropriate Vysis probe protocol.

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