Calcein Staining Protocol for adult sticklebacks

REAGENTS:

STEPS:

1. Make 10 mg/ml (100X) Calcein solution in dH₂0. Cover this solution with tin foil. I have been storing this at 4 C for a month and it is still fine.
2. Place adult fish in a beaker of fish tank water. (We use .35% Instant Ocean formula in dH₂0.) Since calcein binds calcium phosphate, it is important not to use water that contains that salt). I put one fish in 100 mls of fish tank water, but you can probably fit up to 5 fish in there. Add 1 ml of the 100X solution.
3. Cover beaker with foil and leave for 3-4 hours. If you are in a rush, the fish will be stained within 1 hour, but it might be difficult to see all of the plates, especially the most anterior plates. I left one fish swimming in the solution overnight and the staining looked great.

Calcein is a fluorescein-iminodiacetic complex that fluoresces green when combined with calcium. Examination for fluorescence can be carried out under UV microscopy in the usual manner for green fluorescence. We use a Leica dissecting microscope with a mercury bulb UV light source. The filters are the same for observing GFP. Any fluorescent microscope that you can visualize GFP with should work for calcein.

Example images are found below.