3.3.2. Fixing honey bee gut samples from preserved bees

  1. If the honey bees have been preserved in absolute ethanol or acetone, the tissue has to be softened before dissection. Achieve this by soaking and dissecting the specimen in aqueous alcohol (usually 70-80% ethanol), then wash the gut sample once more with absolute ethanol just prior to fixation. (See 3.3.2 for working with fresh samples.)
  2. Fix the samples by soaking them in Carnoy’s fixative (ethanol: chloroform:acetic acid = 6: 3: 1 [v/v]) at room temperature, overnight. Tissues can be exposed to Carnoy’s fixative for longer time periods if autofluorescence from tissue needs to be reduced further.  However, this is not typically necessary for honey bee gut tissues.
  3. Then wash with absolute ethanol at least three times for 20 min at room temperature.