Staining three-spined sticklebacks preserved in ethanol

The strategy we favor is to throw the fish in 10% formalin from ethanol for 2 weeks (you'll have to dilute the formaldehyde if you get it from stores, 1:10 formaldehyde:water, but there should be some already diluted in the cupboards). After 2 weeks, rinse the fish in water for 24 hours, then follow the normal protocol for formalin-preserved fish (jump to Staining step below).

Warning It is all but impossible to extract DNA from fish that have been subjected to this staining treatment, even fish that have never so much as sniffed formalin.

Alternatively, if you wish to avoid formalin altogether, use the following dilutions. The fish need to be transferred gradually to a water-based solution, otherwise the little bodies fly apart. From 95% ethanol, transfer to 70% ethanol for 24 hrs; 50% ethanol for 24 hrs; 20% ethanol for 24 hrs; tap water 24 hrs.

Staining

1% KOH = 1 gram/100 ml water to which you add enough Alazarin Red powder to turn the solution Purple ("Welch's Grape Juice" colour). Pour rinse water through strainer, replace with Alazarin Red solution. Stain for 24 hours (depending on size of fish, etc.).

Note: Katie Peichel reports success staining small fish in 0.004% alizarin red without KOH.

Rinsing

Rinse fish well with water, up to 24 hours. This will usually clear away any stain picked up by non-bony tissues. To dispose of Alazarin Red solution, neutralize with vinegar (check pH) and pour down drain with water flowing.

Storing

Store rinsed fish in 40% isopropyl alcohol (available in fish museum).

Conclusions

Fish are now ready to be measured or kept in storage. If they are going to be in storage for a long time, it is recommended that you check fluid levels in jars every 6 months or so.