Which is the most complex step of FISH?

The most complex step of FISH is during hybridization. The hybridization process typically requires specific temperatures and strict buffer conditions to promote the specific binding of the probe to its target sequence while minimizing non-specific binding. Additionally, probes must be carefully designed to ensure specificity for the target sequence. Factors such as probe length and GC content can influence hybridization efficiency. If the probe is already fluorescent, the site of hybridization can be directly detected. However, if the probe is not fluorescent, an additional step may be required to visualize the hybridized probe.