Paraffin Pretreatment 3

 

Short version protocol should not replace the package insert.

Intended Use
To prepare paraffin-embedded tissue sections fixed on positively charged slides for use in fluorescence in situ hybridization (FISH*) with Vysis Multi-color DNA FISH probes. 

Summary and Principle
The procedure that follows has been designed to maximize tissue processibility and permeability for FISH when using Vysis Multi-color DNA FISH probes (e.g., ProVysionTM Multi-colorProbe, Order No. 32-131090). This pretreatment assay will help minimize heavy particulate background after DNA probe hybridization, which may sometimes occur in specific tissue types.
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Sample Slides Preparation
Note: Use samples that were fixed in formalin for between 24-48 hours.

  1. Cut 4 - 5 µm thick paraffin sections using a microtome.
  2. Float the sections on a purified (i.e., triple distilled) water bath at 40ºC.
  3. Mount the section on a positively charged slide.
  4. Air dry the slides and bake overnight at 56ºC.

Deparaffinizing Slides

  1. Immerse slides in Hemo-De for 5 minutes at ambient temperature.
  2. Repeat step one twice, using fresh Hemo-De each time.
  3. Dehydrate slides in 100% EtOH for 1 minute at ambient temperature. Repeat.
  4. Air dry slides for 2-5 minutes, if desired.

Slide/Protease Pretreatment

  1. Immerse slides in ambient 45% formic acid/0.3% hydrogen peroxide for 15 minutes.
  2. Immerse slides in purified water for 3 minutes. Remove excess water by blotting the edges of the slides on a paper towel.
  3. Immerse slides in Pretreatment Solution at 80°C for 10 minutes.
  4. Rinse in fresh purified water for 3 minutes.
  5. Incubate in 37°C Protease Working Solution for 10 minutes. Measure 50mL (EXACT) of Protease Buffer III and place in a 37±1°C water bath. Ensure that the temperature of the buffer is 37±1°C, and prior to use. Prepare the Protease Stock Solution by adding 9.0 mg (one tube) protease to 515 µL of purified water.

    To make the Protease Working Solution
    Pipet 100 µL of Protease Stock Solution into the (warmed and carefully measured volume) 50 mL bottle of Protease Buffer III. Freeze the remaining Protease Stock Solution.
  6. Stop the reaction by incubating in Protease Stop Solution for 3 minutes.
  7. Rinse slides in purified water for 3 minutes.
  8. Air dry slides for 2-5 minutes.
  9. Proceed with the appropriate Vysis probe protocol.

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