PARAFFIN PRETREATMENT

PARAFFIN PRETREATMENT image
PARAFFIN PRETREATMENT image
PARAFFIN PRETREATMENT image

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 probes.

Reagents Provided

REAGENT QUANTITY COMPOSITION STORAGE
Pretreatment Solution 5 x 50 mL Sodium Thiocyanate (NaSCN) 2 to 25 °C
Protease Buffer 5 x 50 mL NaCl, pH 2.0 2 to 25 °C
Wash Buffer 5 x 250 mg 2X SSC, pH 7.0 2 to 25 °C
Protease 5 x 25 mg 2500 - 3000 U/mg protease, lyophilized -20 °C


Preparing Specimen Slides (optional)

Note: Use specimens 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 40 °C.
  3. Mount a section on a positively charged slide.
  4. Air dry the slides.
  5. Bake the slides overnight at 56 °C.

Deparaffinizing Slides

  1. Immerse slides in Hemo-De for 10 minutes at ambient temperature.
  2. Repeat step 1 twice using new Hemo-De each time.
  3. Dehydrate slides in 100% EtOH for 5 minutes at ambient temperature. Repeat.
  4. Air dry slides or place slides on a 45 - 50 °C slide warmer for 2-5 minutes.

Pretreating Slides

  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. Two slides can 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. 
  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

  1. Remove slides from the second jar of Wash Buffer.
  2. Remove excess buffer by blotting the edges of the slides on a paper towel.
  3. Immerse slides in Protease solution at 37 °C for 10 minutes. (Ensure that the temperature of the buffer is 37 ±1 °C prior to adding 25 mg (one tube) protease.
  4. Immerse slides in Wash Buffer for 5 minutes. Repeat using the second jar of Wash Buffer.5. Dry slides on a 45 - 50 °C slide warmer for 2-5 minutes.

Fixing the Specimen

  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.
  4. Proceed with the appropriate Vysis probe protocol.