Alizarin Red Staining

From SticklebackWiki

Last Updated: May 18, 2005
Prepared by Mark Currey; adapted from similar protocols from Bonnie Ullmann and William Cresko

Procedure

1. Fix: 4% PFA or 10% formalin, fix over night

   (If fish have been kept in 100% Ethanol (eg: for DNA extraction) fix in 20% formalin 48hr)

2. Wash: 3 x 10 min with Distilled Water
3. Bleach: 3%H2O2/0.5%KOH until pigment is gone on body and eyes are light brown
4. Wash: 0.5% KOH 2 x 5 min
5. Stain: 10ml Alizarin Red stock into 1000ml 1% KOH.

   Leave fish in solution until bones are stained being careful not to over-stain other tissues.**

6. Destain: 1% KOH over night or longer at room temperature
7. Storage: Short term: 50% isopropanol or 0.5% KOH, Glycerol

   Long term: 100% Glycerol with a few thymol crystals added to prevent mold spores.

Alizarin Red Stock Solution

Stock Alizarin Red (0.5% alizarin red S): To make 50 mls add 0.25g alizarin red S powder to 50 ml water.

**This is a concentrated solution so that care will be needed to not over-stain. To avoid this you can use 5ml of stock solution in 1000ml 1%KOH for a longer period of time.

Bleach

• 3% H202/0.5%KOH: Mix just before using: equal parts 6%H202 & 1%KOH