PROTOCOL 001: Staining Zebrafish Larval Cartilage with Alcian Blue
4% PFA IN PBS
Alcian blue 8GX (Sigma A3157)
Trypsin (Sigma T-4799)
PBS + Tween 20 (0.1% v/v)
Fix larvae in 4% PFA overnight at 4 ¡C in 1.5 ml microfuge tubes.
Remove PFA and rinse 2X in PBS + Tween 20. At this point, larvae can be stored in 100% MeOH at 4 ¡C.
Make stain: 0.1 mg/ml in 4:1 EtOH/Acetic acid – IMPORTANT: make on day of use. Discard unused stain.
Add 1 ml of stain solution. Stain overnight at RT with or without rocking.
Remove stain. Rehydrate larvae: 90% EtOH ¨ 50% EtOH ¨ 30% EtOH for 2 minutes per step at RT. Remove EtOH.
Make bleaching solution: 1% H2O2/1% NH3 (make with 30% H2O2 and concentrated ammonium hydroxide in fumehood!)
Bleach larvae with H2O2/NH3 solution for 20' at RT. Remove bleach solution. Note: larvae may be sticky and using a Pasteur pipette will circumvent this problem.
Make trypsin solution: 50 mg/ml in 30% Na borate made from saturated sodium borate – IMPORTANT: make on day of use. Discard unused enzyme.
Add 1 ml Trypsin/Na borate solution. Rock at RT for ² 2 h.
Remove Trypsin/Na borate solution. Rinse larvae 3 – 5X in PBS + Tween 20.
Source: Zf3, p72