Whole-mount method for staining bone and cartilage

(version 1.0; 01/27/04)

Mike Shapiro

This protocol is optimized for small sticklebacks, but can be used for virtually any small vertebrate specimen.

1. Fix specimens in 10% neutral-buffered formalin (NBF) at room temperature. Small fry may be fixed overnight, larger adults (SL Ñ 50 mm) for up to one week. VARIATION: if only cartilage staining in very small fry is desired, you can fix in Dentπs fixative (20% dimethyl sulfoxide in methanol) instead of NBF.

2. Rinse in dH₂O 2X, 10 minutes each wash.

3. Remove entrails of specimens if practicable, being careful not to damage ventral skeletal structures. For embryos, fry, and small juveniles, this step may be eliminated.

4. Wash specimens several times in dH₂O at room temperature (on a rocker, if possible). Adult specimens with long fixation times should be washed overnight, while fry can be washed for a couple of hours. Blot larger specimens on paper towels to remove as much water as possible.

5. Immerse specimens in Alcian blue (20 mg Alcian blue 8GX, 70 ml 100 EtOH, and 30 ml glacial acetic acid; this solution can be stored at 4∫C for several months) until desired intensity of staining of cartilage is achieved. IMPORTANT: CLOSELY MONITOR THE STAINING OF SMALL SPECIMENS. This solution is highly acidic and will quickly dissolve the bone of fry and small juveniles. Excellent cartilage staining of small (SL æ 15 mm) fish can typically be achieved in 1-4 hours.

6. Run the specimens through an ethanol:dH₂O series (3:1, 1:1, 1:3; 1 hour each wash) into dH₂O. Washes can be shortened for small specimens; in general, when the specimen sinks, it has equilibrated. Specimens can be held in 70% EtOH indefinitely. It is also possible to ≥back down≤ from this step into Alcian blue to intensify staining.

7. Wash specimens two times in 30% saturated sodium borate, 5 minutes each wash. Saturated sodium borate solution is made by adding sodium borate powder to water until the solution does not dissolve additional salt. Stir solution overnight, allow undissolved salt to settle, and decant saturated solution.

8. Immerse specimens in trypsin solution (0.25-1% trypsin in 30% sodium borate) until translucent. This step may take 1-2 hours for small embryos and 2 days or longer for larger specimens. For overnight treatment of very small fry, the trypsin concentration may be lowered to 0.12% or less.

9. Remove trypsin solution and wash specimens twice with 2% potassium hydroxide, 5 minutes each wash.

10. Immerse specimens in alizarin red solution (0.001-0.002% Alizarin Red S powder in 2% potassium hydroxide). Thorough staining of bone may take from 8-24 hours.

11. Bleach specimens in 3 parts 0.5% potassium hydroxide to 1 part glycerol to which 30% hydrogen peroxide (50µl of 30% H₂O₂ per 10ml of KOH:glycerol solution) has been added. For larger specimens, bleaching may take several days to complete. Hydrogen peroxide concentration may be lowered (as low as 10µl/10ml) for very small specimens or if interstitial bubble formation is a concern.

12. Transfer specimens through a 0.5% potassium hydroxide:glycerol series (1:1, 1:3, 100% glycerol). Each step should be allowed at least two hours, and specimens should sink prior to transfer into the next step. It is possible to ≥back down≤ from this step back into trypsin solution to increase translucency, or into alizarin red to intensify staining.

13. Specimens should be stored in 100% glycerol. Thymol (several crystals per 5ml vial) or phenol can be added to inhibit bacterial growth.

This protocol is based largely on the following reference:

Dingerkus, G., and L. D. Uhler. 1977. Enzyme clearing of Alcian blue stained whole small vertebrates for demonstration of cartilage. Stain Technology 52: 229-232.