3.2. Preserving insect materials for FISH
Specimen quality can substantially influence the success of FISH experiments. Immediately processed fresh insect specimens represent the optimal material. The tissue should be dissected (see Carreck et al., 2013) in an isotonic solution, such as phosphate buffered saline (PBS) or Insect Ringer’s solution, before being fixed, as this will facilitate the infiltration of reagents into tissue later. Dissection prior to fixation also aids in orienting the specimen for later sectioning.
However, insects are often collected in the field, and thus they cannot immediately be processed. If the insects must be preserved before FISH experiments can be performed, acetone and ethanol can be used as preservatives. Acetone is an amphiphilic organic solvent that deprives water from fresh tissue samples very quickly. This property makes acetone an excellent preservative agent for insects with cuticle exoskeletons that prevent efficient penetration of many aqueous fixative agents (Fukatsu, 1999). However, acetone is not always the best choice for FISH experiments, especially in case of bee gut specimens. The major flaw of acetone is that tissues soaked in it become brittle. This makes the dissection of an intact gut from whole body preservations in acetone extremely challenging, if not impossible. Hence, absolute ethanol is recommended if entire insects need to be preserved for later dissection. However, if the desired tissue can be dissected prior to preservation then acetone is the primary choice.
Use excessive amounts of preservative agent. The total volume of insect tissue should be roughly 10% of the preservative volume. These preservatives are highly volatile and their volume can decline fairly quickly over time. Thus, be sure to check the volume periodically in preserved specimens.