Troubleshoot Processing

FISH is a complex multi-step assay where chemical, physical, and optical variables interact. When an assay fails, the “symptoms” observed under the microscope (e.g., no signal, high background, cross-hybridization) can be traced back to a specific failure point in the protocol. The laboratory scientist uses a troubleshooting algorithm to identify the root cause and determine if the slide can be salvaged (re-processed) or if the assay must be repeated from the beginning

Problem 1: No Signal (The “Black Slide”)

• Symptom: DAPI nuclei are visible, but no Red/Green probe signals are seen
• Root Cause Analysis
  • The Probe: Was the probe actually added? (Human error). Was the probe expired or degraded (left in light)?
  • Denaturation (The Prime Suspect)
    • Under-Denaturation: Check the DAPI. If nuclei are shiny/refractile, the DNA never melted. The probe bounced off
    • Fix: You can re-denature this slide. Wash off the DAPI, re-apply probe, and denature at a higher temp (e.g., \(+2^\circ\text{C}\))
  • Wash Stringency
    • Over-Washing: Was the post-wash too hot (\(>74^\circ\text{C}\))? This strips the specific signal
    • Fix: Cannot save this slide. Must repeat with a new slide and lower wash temp
  • The Microscope: Is the shutter open? Is the fluorescence lamp (bulb) dead or old (>200 hours)?

Problem 2: Weak Signal (Faint/Grainy)

• Symptom: Signals are visible but require straining the eyes. They fade instantly (photobleaching)
• Root Cause Analysis
  • Specimen Quality: “Old” slides (stored for years) lose DNA integrity
  • Drift: The optical filters or lamp alignment may be off
  • Re-Processing: If a slide was processed twice, the DNA degrades, leading to weaker signals each time
  • Fix
    • Increase the hybridization time (e.g., 48 hours instead of 16)
    • Use a fresh probe aliquot
    • Check the Antifade (expired antifade allows rapid bleaching)

Problem 3: High Background (The “Green Haze”)

• Symptom: The entire nucleus or the glass slide glows green/red. It is hard to distinguish true dots from the noise
• Root Cause Analysis
  • Wash Stringency (Under-Washing): The post-wash was too cold or too salty. The non-specific probe wasn’t removed
    • Fix: You can re-wash this slide. Remove coverslip, wash again at the correct temp, and re-counterstain
  • Drying: If the slide dried out during hybridization (chamber wasn’t humid), the probe binds irreversibly to the glass
    • Fix: Unsalvageable. Repeat
  • Cytoplasm: Heavy cytoplasm traps probe
    • Fix: Treat the next slide with Pepsin to digest cytoplasm before hybridizing

Problem 4: Cross-Hybridization (Non-Specific Binding)

• Symptom: In addition to the 2 bright target signals, there are many fainter signals on other chromosomes
• Root Cause Analysis
  • Cot-1 DNA Failure: The repetitive sequences (Alu repeats) in the probe were not blocked
  • Probe Concentration: Too much probe was used
  • Homology: The target gene has a “pseudogene” elsewhere in the genome with similar sequence
• Fix: Increase the stringency of the post-wash (higher temp) to strip off the weaker cross-hybridized binding while keeping the strong specific binding

Problem 5: Autofluorescence (Glowing Debris)

• Symptom: Debris or cells glow yellow/orange (visible in both Red and Green channels)
• Root Cause Analysis
  • Biological: Red blood cells, necrosis, or collagen naturally fluoresce
  • Fix
    • During Analysis: Switch between filters. True signal is only visible in one channel (e.g., Red). Autofluorescence is visible in all channels
    • During Prep: Use a fixative wash (Carnoy’s) to lyse RBCs more thoroughly. For tissue, use specialized “Quenching” reagents (e.g., Sudan Black) to mask autofluorescence

Problem 6: “Doughnut” Signals

• Symptom: Signals look like hollow rings or halos
• Root Cause: Over-Denaturation. The heat was too intense, causing the DNA in the center of the spot to disintegrate or “bubble.”
• Fix: Lower the denaturation temperature by \(2^\circ\text{C}\) for the next run