FFPE Tissue Sections

Formalin-Fixed Paraffin-Embedded (FFPE) tissue is the standard preservation method for solid tumor pathology (breast, lung, colon, melanoma). Unlike fresh cell suspensions, these tissues are biologically “locked” in a crosslinked matrix of proteins and wax. Processing FFPE slides for FISH requires a rigorous Pretreatment protocol to undo this preservation just enough to let the probe in, without destroying the tissue architecture. The workflow is significantly longer and more aggressive than standard cytogenetics

Step 1: Deparaffinization (Dewaxing)

Paraffin wax is hydrophobic (repels water). Since FISH probes are dissolved in water-based buffers, the wax must be completely removed

• Reagent: Xylene (or safe alternatives like CitriSolv)
• Protocol: The slides are soaked in Xylene washes (typically 2–3 changes for 10 minutes each) to dissolve the wax
• Rehydration: The slides are then passed through a graded alcohol series (100% \(\rightarrow\) 85% \(\rightarrow\) 70% Ethanol \(\rightarrow\) Water) to replace the solvent with water

Step 2: Pretreatment (Heat-Induced Epitope Retrieval)

Formalin fixation creates methylene bridges (crosslinks) between proteins and DNA. These crosslinks cage the DNA, preventing probe access

• Mechanism: Boiling the slides in a specialized acidic or basic buffer breaks these crosslinks
• Protocol: Slides are incubated in Pretreatment Solution (e.g., Sodium Thiocyanate or Citrate Buffer) at \(80^\circ\text{C}\)–\(98^\circ\text{C}\) for 10–30 minutes
  • Balance: Too little heat = No signal (DNA locked). Too much heat = Tissue falls off the slide or DNA degrades

Step 3: Protease Digestion (Pepsin)

Even after removing wax and crosslinks, the nucleus is still surrounded by a dense mesh of cytoplasmic proteins and membranes

• Mechanism: A proteolytic enzyme (usually Pepsin or Proteinase K) digests the protein scaffold
• Protocol: Slides are incubated in Pepsin solution at \(37^\circ\text{C}\) for 10–60 minutes
• The Critical Check: This is the most variable step. Different tissue types (breast vs. bone) digest at different rates
  • Under-digested: Nuclei are covered in cytoplasm. DAPI stain shows undefined boundaries. FISH signal will be absent or surface-only
  • Over-digested: “Ghost” nuclei. The nuclear structure collapses, and DNA morphology is lost. DAPI is weak. The tissue looks like “Swiss cheese.”
• Correction: Labs often optimize digestion times for each tissue type (e.g., Breast = 15 mins; Lung = 20 mins)

Step 4: Post-Fixation (Optional)

Some protocols include a brief re-fixation in 10% Formalin after digestion to stabilize the fragile exposed nuclei before the violent heat of denaturation

Step 5: Co-Denaturation & Hybridization

Once the tissue is “opened,” the probe is applied

• Denaturation: FFPE DNA is robust. It typically requires higher denaturation temperatures than blood cells (e.g., \(80^\circ\text{C}\)–\(85^\circ\text{C}\) vs. \(73^\circ\text{C}\)) to melt the DNA strands
• Hybridization: Standard overnight incubation at \(37^\circ\text{C}\)

Unique Challenges of FFPE FISH

• Sectioning Artifacts
  • Truncation: Nuclei are sliced by the microtome. You might only have half a nucleus on the slide. This leads to False Monosomy (signal loss)
    • Correction: Laboratory scientistsscore only intact, non-overlapping nuclei
  • Overlap: In thick sections (\(>5 \mu\text{m}\)), nuclei stack. This leads to False Polysomy (signal gain)
• Auto-fluorescence: Connective tissue (collagen, elastin) and necrotic tumor areas glow green/red. The analyst must distinguish distinct sharp probe signals from the diffuse glowing background
• Probe Selection: Because truncation causes false deletions, diagnostic cut-offs for deletions (e.g., PTEN loss) are set much higher in tissue (e.g., >20% of cells) compared to liquid suspensions