Molecular
Molecular

Paraffin Pretreatment 2

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 permeability for FISH when using Vysis Multi-color DNA FISH probes.

Reagents Provided in Kit   Kit Order #32-801210  For Laboratory Use

Reagent Quantity Composition Storage
Pretreatment Solution 5 x 50 mL Sodium Thiocyanate (NaSCN) 2 to 25 °C
Protease Buffer II 5 x 62.5 mL 0.2 N HCl, pH 1.0 2 to 25 °C
Protease 5 x 250 mg 2500 - 3000 U/mg protease, lyophilized -20 to 8 °C

Materials Required but not Provided

  • Absolute ethanol (EtOH)
  • Hemo-De clearing agent (Scientific Safety Solvents Cat. #HD-150)
  • Purified water (distilled or deionized)
  • Coplin jars (16 slide / 8 slots capacity)
  • 37 °C and 80 °C water baths (one at 73°C for the probe assay)

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 a section on a positively charged slide.
  4. Air dry the slides.
  5. Bake the slides overnight at 56ºC.

Deparaffinizing Slides

  1. Immerse slides in Hemo-De for 5 minutes at ambient temperature.
  2. Repeat step one (1) 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 Pretreatment

  1. Immerse slides in Pretreatment Solution at 80ºC for 10 minutes. If necessary, two slides may be placed back-to-back in each slot in the Coplin jar, with one slide placed in each end slot. For the end slides, the side of the slide with the tissue section must face the center of the jar.
  2. Immerse slides in purified water for 3 minutes.

Protease Pretreatment

  1. Remove slides from the jar of purified water.
  2. Remove excess water by blotting the edges of the slides on a paper towel.
  3. Immerse slides in Protease solution at 37ºC for 15 minutes. (Ensure that the temperature of the buffer is 37±1ºC prior to adding 250 mg (one tube) protease. If necessary, two slides may be placed back-to-back in each slot in the Coplin jar, with one slide placed in each end slot. For the end slides, the side of the slide with the tissue section must face the center of the jar.
  4. Immerse slides in purified water for 3 minutes.
  5. Air dry slides for 2-5 minutes.

Fixing the Sample

Note: Fixation of the sample is performed to minimize tissue loss during sample denaturation. This procedure is highly recommended when processing samples in a denaturatrion bath format.

  1. Fill one (1) Coplin jar with 50 mL of 10% buffered formalin. Fill three (3) other Coplin jars with 50 mL of 70% ethanol, 85% ethanol and 100% ethanol in each.
  2. Immerse the slides in 10% buffered formalin at ambient temperature for 10 minutes.
  3. Immerse the slides in purified water for 3 minutes.
  4. Air dry slides.
  5. Proceed with the appropriate Vysis probe protocol.
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