Vertebrate Clearing and Staining - 'VCaS' Protocol

De-gut and de-scale/skin (optional, but recommended)

These processes should not be carried out if the specimen are too fragile or if either of the two features are of interest to the study.

If the specimen is stored in solution (e.g. formaldehyde, ethanol, isopropyl alcohol, etc.), rinse the specimen by draining the fluid according to the chemical-specific laboratory waste requirements.

Rinse the specimen by gently adding and pouring out

2 to 3 times.

Label sterile containers with sample or identification assigned to specimen.

Remove the specimen and place into a clean plastic or glass container large enough for the specimen to be completely submerged and flat on its side.

If cartilage staining is desired, go to Step 4 . If only the bone structure is of interest, skip to Step 5 and start the clearing process.

This step is used to stain cartilage. Cartilage is stained with

Place the specimen into a solution of 800 ml of 95% , 200 ml of 100% (pH 2.5), and 1 gram of .

Remove from solution when it reaches desired staining (i.e. desired intensity of blue color).

After staining, dispose of the used Alcian solution according to laboratory waste requirements or return to a container for reuse. Specimen should then be gently rinsed using water to remove any extra stain.

Place the specimen into a clean plastic or glass container large enough to completely submerge specimen during clearing.

Trypsin enzyme is used to begin the clearing process by breaking down (bleaching) proteins in the flesh of the specimen. This allows for viewing of internal structures.

To begin the clearing process, mix 3:7 ratio of saturated solution and (e.g. 150ml saturated Sodium borate solution : 350ml ddH2O) in a clean beaker until solution is clear. These can be combined using a magnetic stirrer/hot plate. If necessary, solution can be further warmed using the hot plate setting.

In the same beaker, mix the sodium borate solution with Trypsin. Mix with a ratio of approximately 50g of to 150ml of sodium borate solution.

Pour Trypsin solution over the specimen until it is fully submerged.

The solution will run out of active Trypsin every two to three days depending on the amount used. Completely replace mixture, including both sodium borate solution and trypsin powder as needed. When swapping out the solutions, try to keep specimen as stagnant as possible. This can be done by gently pouring out solution while holding specimen in place with plastic spoon or other non-metal object.

After processes are complete, remove the specimen from the final solution and dispose of the used Trypsin solution. Specimen should then be gently rinsed using to remove any unwanted remnants.

Prepare hydrogen peroxide solution.

To do so, combine 150mL of with 2-4 drops of (or an equivalent amount of a lower concentration H₂O₂) in a container. Mix with a glass stirring rod.

Place specimen in hydrogen peroxide solution for no more than 24 hours to prevent decomposition.

Remove specimen from hydrogen peroxide solution and rinse with to remove any remaining .

The specimen is now ready for staining.

Prepare Alizarin red solution.

To do so, a 0.5% solution must be created by combining 5 grams of and 1000 mL of in a container. Mix with glass stirring rod.

Once 0.5% solution is created, mix with powder until completely dissolved and desired color is reached.

Place specimen in the Alizarin red solution and watch closely for change in hue. Remove from container once specimen has reached desired saturation.

When desired color is reached, remove the specimen from Alizarin red and place in plain 0.5%

solution to remove excess Alizarin red. Replace

solution periodically over a 24-48hr period until the solution remains clear.

Take the specimen through three different concentrations (2:1, 1:1, 1:2) of (KOH) : solutions to fully clear the tissue.

First, place the specimen into a 2:1 : solution with enough liquid to fully submerge the specimen.

Carefully remove specimen from the first solution and place into a 1:1 : solution.

Carefully remove specimen from the second solution and place into a 1:2 : solution.

Carefully remove specimen from the third solution and place it into 100% .

The cleared and stained specimen can be preserved in a container of 100% .

Optionally, a couple drops of may be added to the glycerine the final specimen will be stored in to aid in any final bleaching of dark spots. One crystal of may also be added to prevent mold growth.