3.4.2. FISH controls
Control experiments are necessary to validate the specificity of the probes and to discriminate between the fluorescent signals of probes and autofluorescence of insect tissues. All three of the following control experiments should be conducted, because they are crucial for verifying any FISH results obtained.
- No probe control. Conduct hybridization without the fluorescent probe(s) to examine the levels of autofluorescence from the tissues.
- Competitive suppression control. Conduct hybridization with an excess amount (usually 50x) of an unlabelled oligonucleotide using the same sequence as a fluorescent probe to observe sequence-specific, competitive suppression of the hybridization signals.
- RNase A control. Prior to hybridization, treat the tissue sections with 10 µg/mL of RNase A in PBSTx at 37˚C for 30 min. Wash the section with PBSTx and then conduct hybridization as usual. The purpose of this control is to verify that the probe specifically binds to RNA molecules.