Clearing And Staining For Larval Fish Cartilage And Bone
(Source: the Parichy Lab from Zebrafish Book 5th Edition)
1. Fixation
Fix fish in 4% paraformaldehyde in PBS for 2 x overnight at 4°C.
2. Dehydration
For larvae less than 20 mm SL:
a. Wash 2 x 5 minutes in PBS.
b. Dehydrate in 50% EtOH / 50% nano water for 24 hours.
c. Dehydrate in 100% EtOH for 24 hours.
For larvae more than 20 mm SL:
a. Wash 2 x 5 minutes in PBS.
b. Dehydrate in 50% EtOH / 50% nano water for 48 hours.
c. Dehydrate in 100% EtOH for 48 hours with one intermediate solution change.
3. Staining cartilage
Cartilage staining solution:
For 100 ml final volume:
70 ml 100% EtOH
30 ml acetic acid
20 mg Alcian blue
a. Transfer specimens to staining dish.
b. Incubate in cartilage staining solution for 24 hours at room temperature with mild agitation.
4. Neutralization
Wash in saturated sodium borate solution for 9-12 hours.
5. Bleaching (optional)
Bleaching solution:
For 100 ml final volume:
15 ml 3% hydrogen peroxide
85 ml 1% potassium hydroxide
Incubate in bleaching solution for:
20 minutes for specimens less than 20 mm SL
40 minutes for specimens more than 20 mm SL
6. Trypsin digestion
Digestion solution:
For 100 ml final solution:
35 ml saturated sodium borate
65 ml ddH2O
1 g trypsin
Incubate in solution at room temperature with mild agitation until specimens are 60% clear.
7. Staining bone
Bone staining solution:
1% potassium hydroxide with
0.1 g/100 ml Alizarin red
Incubate in bone staining solution for 24 hours at room temperature with mild agitation.
8. Destaining
For specimens less than 20 mm SL:
Incubate in trypsin digestion solution for 40-48 hours.
For specimens more than 20 ml SL:
Incubate in trypsin digestion solution until specimen is clear and solution remains unstained. Change to fresh solution every 2 days.
9. Preservation
a. Transfer to 30% glycerol/70% of 1% potassium hydroxide. Incubate 2-3 days at room temperature with mild agitation.
b. Transfer to 60% glycerol/40% of 1% potassium hydroxide. Incubate 2-3 days at room temperature with mild agitation.
c. Transfer to 100% glycerol with thymol.
Reference
Adapted from: Potthoff, T. (1984). Clearing and staining techniques. In: Ontogeny and Systematics of Fishes (based on an international symposium dedicated to the memory of Elbert Halvor Ahlstrom). H.G. Moser, W.J. Richards, D.M. Cohen, M.P. Fahay, A.W. Kendall, Jr., and S.L. Richardson, eds. Lawrence, KS, Special Publication 1, American Society of Ichthyologists and Herpetologists, Allen Press, pgs. 35-37.