Utilize the DAPI filter to review the shape of the nuclei. DAPI stains the DNA in the nuclei, and when examining the slide with a fluorescent microscope using the DAPI filter, the nuclei will appear blue, as shown in the image. The borders of the nuclei should be distinct so that you can discern each cell. The nuclei should have a homogeneous blue color and not have a "hole" or lighter area in the center. If the cells appear lighter or have a hole in the center, the cells have been over-digested.
Scan the slide on each filter that corresponds to a probe. Look for well-defined signals. A triple bandpass filter will allow you to see the DAPI-stained nuclei with the signals within them. Sometimes, reviewing the triple bandpass filter to determine the cell's borders regarding the signals is helpful. Actual signal counting is usually easier using dual filters. Single filters are sometimes used to determine if signals are overlaying each other.
