What should be done if high non-specific background is observed on a postwashed FISH slide?

When encountering high non-specific background on a post-washed FISH (Fluorescence In Situ Hybridization) slide, increasing the post-wash temperature can help enhance the specificity of the probe binding. Higher temperatures can reduce the binding of the probe to non-target regions by promoting dissociation of non-specifically bound probes, thereby decreasing background fluorescence. This enables clearer visualization of the specific hybridization signals.

While other options may seem reasonable in different contexts, they do not directly address the issue of non-specific background in the same effective manner as adjusting the post-wash temperature. For instance, reducing the dye concentration might not necessarily eliminate non-specific binding, as some probes can still bind indiscriminately at lower concentrations. Changing the type of probe used may also be a solution but is more drastic and might not be required if temperature adjustments can effectively resolve the background issue. Reducing exposure time to the light source could potentially decrease the background signal, yet it risks underexposing the specific signals that are of interest, leading to a loss in overall clarity and detectability. Thus, increasing the postwash temperature is the most direct and effective approach to reducing high non-specific background in FISH analysis.