Imaging ROS Production Using DHE

  1. Collect and dissect larvae in warm 1X PBS.
    CRITICAL STEP Do not use ice-cold PBS, as this may inhibit respiration and thus interfere with ROS production.
    CRITICAL STEP Do not dissect in Schneider's medium, as the presence of primary amines from the amino acids it contains can lead to extracellular   hydrolysis of the dye. In addition, it is important to remove as much extraneous tissue as possible. However, in this case tissue should be dissected and incubated in Schneiders medium to allow optimal respiration.
  2. Reconstitute dye right after dissection and immediately before use.
    CRITICAL STEP Reconstituted dye solution should appear slightly pink in color, a more intense color such as purple may be indicative of autooxidation of the dye.
  3. Dissolve 1 ul of the reconstituted dye ( i.e in DMSO) in 1ml of schneiders medium (a final concentration of approximately 30uM: add 100 ul of DMSO). Vortex to evenly disperse the dye.
    CRITICAL STEP Vortexing for about 15 to 30 seconds is usually optimal. Excessive vortexing may hasten decomposition of the dye, as it is subject to hydrolysis; on the other hand, shorter vortexing times may result in incomplete dispersion of the dye, resulting in the deposition of colloids on the tissue.
  4. Incubate the tissue with the dye for 3 to 7 minutes in a dark chamber, on an orbital shaker at room temperature.
  5. Perform three 5-minute washes in schneiders medium in a dark chamber, on an orbital shaker at room temperature.
  6. (Optional) Fix slightly for 4 to 8 minutes in 7% formaldehyde in 1XPBS.
    CRITICAL STEP When first examining a tissue for ROS production using DHE,
    it is important to go through the protocol without fixing to get an accurate indication of live ROS production. However, the mild fixation step described here facilitates analysis of ROS production in GFP marked clones, as completely unfixed GFP samples tend to produce clones with fuzzy boundaries. The length of the fixation is crucial, and may have to be determined empirically for the tissue of interest and particular variant of GFP used: too little fixation will produce fuzzy GFP boundaries but will significantly retain ROS signal, excessive fixation will produce clear GFP boundaries but will compromise ROS signal.
  7. Rinse once in 1X PBS right after fixation.
  8. Mount immediately in vectashield.
  9. Capture images immediately using a confocal microscope.