1. Prepare a 0.01Â % ARS solution, using water from the system in which fish were previously maintained (system water or embryo medium) |
 1.1. A 5× concentrated solution (0.05 %) can be prepared with distilled water, then diluted in embryo medium or system water to 0.01 % working solution before use |
 1.2. Adjust pH to 7.4 with KOH solution |
 1.3. Keep solution in the dark when storing |
2. Transfer fish to ARS solution |
 2.1. Adult specimens can be transferred with fish nets |
 2.2. Larval specimens can be transferred using Pasteur pipettes |
3. Stain for 15Â min with ARS solution |
4. Rinse at least 3 times for 5Â minutes in embryo medium or system water |
 4.1. Substitute staining solution with new embryo medium or system water, or transfer fish into new containers, as described in points 2.1. and 2.2. |
5. Perform image analysis and photograph acquisition |
 5.1. Anaesthetize specimens with up to 0.6 mM MS222 |
 5.2. Accommodate specimens for imaging (e.g., Petri dishes, glass-bottom dishes, excavated slides) |
 5.3. Use fluorescent microscope or stereomicroscope, depending on the desired magnification, coupled to the appropriate fluorescent filter |
 5.4. Image under green fluorescent light (510–550 nm) |
6. Recover fish from anaesthesia, by transferring them to new embryo medium or system water |