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Cytogenetics

Counterstain

The counterstain is the final chemical step in the FISH protocol. After the probe has hybridized and the non-specific noise has been washed away, the slide essentially contains invisible DNA spots. To visualize the context - the nucleus or the chromosomes - a fluorescent dye that binds generally to DNA is applied. This provides the “map” upon which the probe signals (the “landmarks”) are located

DAPI (4’,6-diamidino-2-phenylindole)

DAPI: is the universal counterstain used in clinical cytogenetics

  • Mechanism: It binds strongly to A-T rich regions in the minor groove of the DNA double helix
  • Fluorescence: When excited by ultraviolet light (358 nm), it emits a bright Blue fluorescence (461 nm). This blue contrasts sharply with the typical probe colors of Green (FITC), Red (Texas Red/Rhodamine), and Aqua, allowing for easy multi-color analysis
  • Concentration
    • DAPI II (High Concentration): Used for metaphase spreads to produce a “G-banding like” pattern (DAPI-banding). The differential binding to AT-rich heterochromatin creates a distinct banding pattern that allows the analyst to identify chromosomes even without Giemsa
    • DAPI I (Low Concentration): Often used for interphase nuclei to avoid overpowering the probe signals. If the blue is too bright, it can “drown out” faint green signals

Antifade Mounting Medium

DAPI is rarely applied as a pure powder or liquid; it is dissolved in an Antifade Mounting Medium (e.g., Vectashield or ProLong Gold)

  • Photobleaching: Fluorescent dyes are unstable. When exposed to the high-intensity light of the microscope (excitation), they rapidly lose their ability to fluoresce - a phenomenon called photobleaching or “fading.” A green signal might vanish within 10–20 seconds of viewing
  • Protection: The mounting medium contains chemical scavengers (free radical inhibitors) that significantly retard this fading process, allowing the laboratory scientist to analyze the slide for minutes or archive it for months

Application & Storage

  1. Application: A small drop (\(10 \mu\text{L}\)) of DAPI/Antifade is pipetted onto the dry slide. A glass coverslip is gently lowered to spread the medium. Air bubbles must be avoided, as they refract light and create dark voids in the image
  2. Curing: Some media require a “curing” time (15–30 minutes) in the dark to harden slightly before viewing
  3. Storage
    • Darkness: Slides must be stored in light-tight folders
    • Cold (\(4^\circ\text{C}\) or \(-20^\circ\text{C}\)): Cold storage prolongs the life of the fluorophores
    • Archival: While G-banded slides last for decades, FISH slides are transient. The fluorescence degrades over weeks to months even in optimal storage. Digital imaging is the only permanent record

Troubleshooting Counterstain

  • “Blown Out” Blue
    • Problem: The DAPI is so bright it hurts the eyes or obscures the red/green signals
    • Cause: Too much DAPI applied or concentration too high
    • Fix: Blot excess medium or use a lower concentration formulation
  • Weak/Pale Nuclei
    • Problem: Nuclei are barely visible
    • Cause: The Antifade medium has expired (yellowed) or the DAPI was not stored properly (light exposure)
  • Hazy Background
    • Problem: The entire slide glows blue
    • Cause: Excess DAPI was not squeezed out or the slide was not washed properly before mounting