PROTOCOL 001: Staining Zebrafish Larval Cartilage with Alcian Blue

(12/2009)

 

REAGENTS

 

4% PFA IN PBS

MeOH

Alcian blue 8GX (Sigma  A3157)

90% EtOH

50% EtOH

30% EtOH

Bleachin solution

Trypsin (Sigma T-4799)

PBS + Tween 20 (0.1% v/v)

 

PROCEDURE

 

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 h.

 

Remove Trypsin/Na borate solution. Rinse larvae 3 – 5X in PBS + Tween 20.

 

 

Source: Zf3, p72