Utilize the DAPI filter to review the shape of the nuclei. DAPI stains the DNA in the nuclei and when reviewing 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 individual 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 the nuclei. Sometimes it is useful to review the triple bandpass filter to determine the borders of the cell in relation to the signals. Actual counting of signals is usually easier using dual filters. Single filters are used at times to determine if signals are overlaying each other.
