American Society for Clinical Pathology (ASCP) Technologist in Cytogenetics certification (ASCP CG) Practice Test

Which counterstain should be used with a fluorescein dye labeled probe during the FISH procedure?

1. Ethidium bromide (blue)

2. Propidium iodide (red)

3. Giemsa stain

4. Methyl green

The correct answer is: Propidium iodide (red)

In the FISH (Fluorescence In Situ Hybridization) procedure, fluorescein dye labeled probes are used to detect specific DNA sequences within cells. When applying a counterstain, it is vital to choose one that does not interfere with the fluorescence of the primary probe. Propidium iodide, which emits red fluorescence, is commonly used because its fluorescent signal can be distinguished from that of fluorescein, which fluoresces green. This allows for effective multiplexing, as you can simultaneously analyze the signals from both the fluorescein probe and the propidium iodide counterstain. The red fluorescence of propidium iodide serves to stain cellular nuclei and assists in visualizing the background, enhancing the clarity of the signals from the fluorescein-labeled probe. Since the choice of a counterstain is crucial for maintaining the integrity of the primary signal while providing helpful visualization, propidium iodide is the ideal choice in this scenario. In contrast, the other options either do not provide the required color contrast necessary for effective analysis or do not serve as appropriate counterstains. For instance, ethidium bromide may interfere with the fluorescein signal due to overlapping fluorescence spectra, while Giemsa stain is typically used for