No Acid No Pain Double Stain

From SticklebackWiki

created from: Walker, M.B., Kimmel, C.B. (2007) A two-color acid-free cartilage and bone stain for zebrafish larvae. Biotechnic and Histochemistry 82, 23-28.

Procedure

1. Fix 2% PFA/1XPBS 1 hr.
2. Rinse 50% ETOH/water 10 mins.
3. Double stain:

   0.04% Alcian Blue/ 0.01% Alizarin Red S/10mM MgCl2/80% ETOH overnight to 5 days.

   Important: Store stock as 0.04% Alcian Blue/10mM MgCl2/80% ETOH. Add Alizarin Red just before use. (20ul 0.5%/ 1ml)

4. Rinse 80% ETOH/10mM MgCl2/water 10 mins to overnight. Cleaner Alcian background with longer rinses.
5. Rinse 50% ETOH/water 5 mins.
6. Rinse 25% ETOH/water 5 mins. (Can fudge here, just rehydrating)
7. Bleach 3% H202/0.5% KOH 10 mins, lids open.
8. Rinse 25% glycerol/0.1% KOH 10 mins to 1 hr.
9. Rinse 50% glycerol/0.1% KOH 10 mins to overnight.
10. Replace with new 50% glycerol/0.1% KOH and store 4°C.

Rock on nutator except for bleach step.