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Sample Pre-treatment: FFPE

  • Applicable samples: Formalin-Fixed Paraffin-Embedded (FFPE)
  • Version: v1.0
  • Last modified: 2026-01-29

What's next?

After completing pre-treatment, proceed to the unified RCA Protocol (Blocking → Hybridization → Ligation → RCA → Post-fix).

Convention

In the following steps, "Jar" denotes a step performed in a separate EasyDip™ Slide Staining Jar previously decontaminated with RNaseZap®. Unless otherwise indicated, all steps are at room temperature (RT).

1. Baking (~16 h)

Goal: Adhere section to slide and stabilize tissue before downstream steps.

  • Attach: One coverslip with a mounted FFPE section onto a microscope slide, taping the edge close to the sample area where the sample name is written.
  • Bake: 56°C for 16 h.

2. Deparaffinization (~1 h)

Goal: Remove paraffin and hydrate tissue through graded solvents.

  • Jar #1: Dewaxing agent (or xylene), 10 min x 2.
  • Jar #2: 100% Ethanol, 10 min.
  • Jar #3: Methanol--acetic acid 3:1 (v/v), 5 min.
  • Jar #2: 100% Ethanol, 5 min.
  • Jar #4: 85% Ethanol, 5 min.
  • Jar #5: 70% Ethanol, 5 min.
  • Jar #6: NFW, 3 min.

3. RNA Retrieval (~1 h)

Goal: Antigen/epitope retrieval to restore RNA accessibility.

  • Jar #7: Sodium citrate 0.01 M pH 6 pre-warmed at 80°C; incubate 45 min at 80°C in a water bath.
  • Jar #6: NFW, 5 min.
  • Jar #5: 70% Ethanol, 5 min.
  • Jar #4: 85% Ethanol, 5 min.
  • Jar #2: 100% Ethanol, 5 min.
  • Air-dry tissue sections at RT.

Note: At this point, sections can be stored dry for several days at +4°C.

  • Assemble Chamber: Cover each tissue section with a PDMS chamber.

4. Rehydration (~20 min)

Goal: Rehydrate tissue for aqueous-based steps.

  • Incubate: 100% Ethanol, 5 min.
  • Incubate: 85% Ethanol, 5 min.
  • Incubate: 70% Ethanol, 5 min.
  • Incubate: NFW, 5 min.

5. Autofluorescence Pre-Quenching (~40 min)

Goal: Reduce autofluorescence with NaBH₄. This step is critical for FFPE samples.

  • Incubate: 1% (wt/vol) NaBH₄ (in 1X PBS) for 30 min.
  • Wash: PBS-Tween 0.05% wash 3 x 2 min.

NaBH₄ Safety

Prepare 10 ml of 1% (wt/vol) NaBH₄ in 1X PBS on ice freshly each time. Use extreme caution when weighing; accidental introduction of liquid into the stock bottle can cause an explosive reaction. Gas bubbles form continuously in the chamber -- aspirate and replenish with fresh solution every 1--2 min during the incubation.

6. Digestion (~20 min)

Goal: Enhanced permeabilization for FFPE tissue (longer than FF).

  • Incubate: 0.04% Pepsin (in 0.1 M HCl) at 37°C for 10 min.
  • Wash: PBS-Tween 0.05% wash 3 x 2 min.

Next step: RCA Protocol (Blocking → RCA)