Table 2.

Bench protocol. Steps of the proposed ARS in vivo staining protocol

1. Prepare a 0.01 % 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 15 min with ARS solution

4. Rinse at least 3 times for 5 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