Alizarin red staining and imaging of zebrafish larvae and adults

Alizarin red staining of zebrafish larvae (7 or 14 dpf)

Reagents

• 25x Tricaine stock solution

• 4% Paraformaldehyde (4% PFA) in PBS

• 10% KOH stock:

o 50g KOH beads

o 500ml dH₂O

• KOH/Triton (2% Triton):

o 440ml (44ml) dH₂O

o 50ml (5ml) 10% KOH

o 10ml (1ml) Triton X-100

• 100% glycerol

• 30% H₂O₂

• Alizarin Red stock (0.5% in H₂O)(C.I. 58005; sigma)

• Triton X-100

Method

1. Euthanize larvae with 25x stock tricaine solution for about 5 min

2. Transfer the larvae to a cell strainer (separate the groups per strainer) with a max number of50-60 per strainer.

3. Wash the larvae with PBS or dH₂O several times to remove the Tricaine.

a. You can place the nets directly on the waste bins while flushing.

4. Place the nets in a 6 well plate and incubate them in 4% PFA for an hour on RT while shaking.

a. Seal the plate with parafilm to prevent evaporation.

b. O/n at 4 °C is also possible.

5. Wash the larvae with PBS or dH₂O several times to remove the PFA.

6. Bleaching, only if pigment is present.

a. Bleaching solution (50ml):

i. 1.5ml 30% H₂O₂

ii. 42.68ml dH₂O

iii. 4.85ml 10% KOH stock

iv. 0.97ml Triton X-100

b. Up to 30 minutes gently shaking at RT

c. Remove the lid of the 6 well plate while bleaching

7. Optional digestion for clearing tissues (such as Yolksack), 45 minutes at RT gently shaking.

a. Digestion solution (50ml):

i. 20ml dH₂O

ii. 30ml 100% Borax (saturated solution)

iii. 1ml Triton X-100

8. Stain using Alizarin.

a. Working solution AR (0.005%) (10ml):

i. 10ml KOH/Triton

ii. 100µl AR stock (0.5% in H2O)

b. Stain for 3 hours at RT while shaking, or o/n at 4 °C

9. Rinse larvae several times with dH₂O

10. Wash in de-staining solution for 30 minutes (if the Otholites are overstained) at RT gentlyshaking.

a. De-staining solution (50ml) 20% glycerol in 0.25% KOH:

i. 38.75ml dH₂O

ii. 1.25ml 10% KOH

iii. 10ml 100% glycerol

11. Bring stained larvae to 100% glycerol, gently increasing concentrations

a. Remove larvae from strainers

b. 30-70-100%

c. 5 min each

12. Store at 4 °C

Reference

Based on: A bone to pick with zebrafish Eirinn W Mackay,Alexander Apschner & Stefan Schulte-Merker. BoneKEy Reports (2013) 2, Article number: 445 (2013) doi:10.1038/bonekey.2013.179