TITLE:                                                                                                                 Vertebrate Clearing and Staining - &#39;VCaS&#39; Protocol
AUTHORS: Sidney E. Ryan, Kara Mia Ehler, Christina M Giudici, Yoel E. Stuart, Emily Y. Hallett, Jacopo Niccolo Cerasoni

[DESCRIPTION]
Small vertebrates can be stained and cleared using a trypsin enzyme, Alcian blue, and Alizarin red stains. The process is completed over several weeks and the final product allows clear viewing of internal structures including bone and cartilage. Cleared and stained vertebrate specimen can be used for educational or scientific purposes. Final products are stored in pure glycerin for an indefinite amount of time and can be viewed over a light box when needed.

[BEFORE_START]
Create an organized inventory of all specimens. Include relevant information to identify specimens (country, site, feature, level, etc.). Assign unique label codes to each specimen (e.g. SITE CODE/YEAR/ID NUMBER). Prepare and print a suitable form for recording observations during protocol.

[GUIDELINES]
In the following protocol references to steps are in italics, e.g. step 3.

[STEPS]
SECTION: Preparation of specimen
1. 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.

 


 
SECTION: Preparation of specimen
3. Label sterile containers with sample or identification assigned to specimen.

 

SECTION: Preparation of specimen
3.1. 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.
SECTION: Enzyme Bleaching
7. 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.
SECTION: Enzyme Bleaching
6. 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. 
SECTION: Enzyme Bleaching
8. Pour Trypsin solution over the specimen until it is fully submerged.

 

 
 

 


SECTION: Enzyme Bleaching
8.1. 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.

 

SECTION: Red Staining
15. Once 0.5%    solution is created, mix with   powder until completely dissolved and desired color is reached.

 

 


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

 

SECTION: Preparation of specimen
2. 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.
SECTION: Preparation of specimen
2.1. Rinse the specimen by gently adding and pouring out 
   2 to 3 times. 

SECTION: Cartilage Staining *Optional*
4. This step is used to stain cartilage. Cartilage is stained with   

SECTION: Preparation of specimen
3.2.  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.
SECTION: Cartilage Staining *Optional*
4.1. Place the specimen into a solution of 800 ml of 95%  , 200 ml of 100%  (pH 2.5), and 1 gram of  .
SECTION: Cartilage Staining *Optional*
4.2. Remove from solution when it reaches desired staining (i.e. desired intensity of blue color).
SECTION: Cartilage Staining *Optional*
4.3. 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.
SECTION: Cartilage Staining *Optional*
4.4. Place the specimen into a clean plastic or glass container large enough to completely submerge specimen during clearing.
SECTION: Final Product
21. The cleared and stained specimen can be preserved in a container of 100%   . 


 


 

SECTION: Red Staining
14. 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.

 

SECTION: Enzyme Bleaching
5. 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.

 


 

SECTION: Final Product
21.1. 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.

SECTION: Glycerin Clearing
19. Carefully remove specimen from the second solution and place into a 1:2   :    solution.
SECTION: Glycerin Clearing
20. Carefully remove specimen from the third solution and place it into 100%  .
SECTION: Glycerin Clearing
18.2. Carefully remove specimen from the first solution and place into a 1:1   :   solution.

SECTION: Glycerin Clearing
18.1. First, place the specimen into a 2:1   :    solution with enough liquid to fully submerge the specimen.
SECTION: Glycerin Clearing
18.  Take the specimen through three different concentrations (2:1, 1:1, 1:2) of   (KOH) :  solutions to fully clear the tissue.

 

SECTION: Enzyme Bleaching
9. 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.

 

 


SECTION: Red Staining
17. 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.


 


SECTION: Hydrogen Peroxide Bleaching
10. 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.

SECTION: Hydrogen Peroxide Bleaching
11. Place specimen in hydrogen peroxide solution for no more than 24 hours to prevent decomposition. 

 


SECTION: Hydrogen Peroxide Bleaching
12. Remove specimen from hydrogen peroxide solution and rinse with    to remove any remaining   .

SECTION: Hydrogen Peroxide Bleaching
13. The specimen is now ready for staining.