3.3.3. Bleaching samples to reduce autofluorescence in the gut tissues

Autofluorescence can be reduced in tissues using hydrogen peroxide (H2O2) (Koga et al., 2009), as described here.  However, autofluorescence is not very strong in honey bee gut tissue and H2O2 treatments produce oxygen bubbles in the lumen causing insufficient penetration of the paraffin. Thus, bleaching with H2O2 is not recommended unless autofluorescence is problematic in your tissues for some reason.

  1. Prepare a solution of H2O2-ethanol using 1 volume of 30% H2O2 and 4 volume of absolute ethanol.
  2. Soak the gut samples in the H2O2-ethanol for several days to several weeks, changing the solution every two to three days. During soaking, the colour of the tissue should become lighter.
  3. Wash with absolute ethanol at least three times for 20 min at room temperature.
  4. The specimen can be kept at -20˚C until use.